Topical macqui berry formula

ABSTRACT

The present invention provides a topical formulation and method of use where the formulation comprises macqui berry or a macqui berry extract containing anthocyanins having a very high oxygen radical absorbance capacity (ORAC). The formulation provides the macqui berry in a stabilized form which includes a glucuronide or glycuronide, a photostabilizing agent, encapsulation, or light—and/or air-blocking packaging.

This Application is a Continuation of U.S. patent application No.15/290,902, filed on Oct. 11, 2016, which Application is a Continuationof U.S. patent application No. 14/480,182 filed on Sep. 8, 2014, whichApplication is a Continuation of U.S. patent application No. 13/226,834filed on Sep. 7, 2011, which Application is a Continuation of U.S.patent application No. 11/525,288 filed on Sep. 21, 2006, whichApplication claims priority pursuant to 35 U.S.C. § 119 from U.S.Provisional Patent Application Ser. No. 60/719,530 filed Sep. 21, 2005,the disclosure of these applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the topical application of compositionscontaining macqui berry or macqui berry extract in a topical formulationfor the prevention and/or treatment of damage to skin, particularly skindamage resulting from chronoaging and photoaging.

BACKGROUND OF THE INVENTION

Skin is subject to abuse by many extrinsic (environmental) factors aswell as intrinsic factors. A common extrinsic factor is exposure toultraviolet radiation. Whether extrinsic or intrinsic, the abuse resultsin skin aging. Skin aging happens in two ways: (1) through the naturalaging process which dermatologists call chronological aging (also knownas chronoaging); and (2) through UV rays in sunlight accelerating theaging process which dermatologists call photoaging. Chronoaging resultsin thinning, loss of elasticity and general degradation of skin. As theskin naturally ages, there is a reduction in the cells and blood vesselsthat supply the skin. There is also a flattening of the dermal-epidermaljunction which results in weaker mechanical resistance. As aconsequence, older persons are more susceptive to blister formation incases of mechanical traumas or disease processes (Oikarinen et al.,Photodermatal. Photoimunmol. Photomed., 7:3-4 (1990)).

By contrast, photoaging, or premature aging, is a process in which theskin changes in appearance as a result of repeated exposure to sunlight.Typically, photoaging occurs in areas of habitual exposure, such as thescalp, face, ears, neck, chest, forearms and hands. The changesassociated with photoaging include elastosis, atrophy, wrinkling,vascular changes (diffuse erythema, ecchymoses, and telangiectasias),pigmentary changes (lentigines, fleckles, and areas of hypo- andhyper-pigmentation), and the development of seborrheic keratosis,actinic keratosis, comedones and cysts.

Antioxidants are useful agents treating the skin from damage caused bychronoaging and photoaging. The most useful antioxidants are those thatprovide the highest capacity to absorb free radicals. The oxygen radicalabsorbance capacity (ORAC) is a measurement of this (Dreher F, MaibachH. Curr Probl Dermatol. 2001; 29:157-64.) Anthocyanins are a type ofantioxidants that generally encompass a class of flavonoid compoundsthat are naturally occurring, water-soluble compounds, responsible forthe red, purple, and blue colors of many fruits, vegetables, cerealgrains, and flowers.

Most anthocyanins have a high ORAC rating compared to other antioxidantsand make them particularly useful for their topical antioxidativeproperties. Additionally, anthocyanins are collagenase inhibitors, whichhelps in the prevention and reduction of wrinkles, increase in skinelasticity, etc., which are caused by a reduction in skin collagen.

For ripe berries, a rich source of anthocyanin antioxidants, there isgenerally a linear relationship between ORAC values and anthocyanincontent. High ORAC ratings are generally defined as greater than 25μmole of Trolox equivalents (TE)/g. In most fruits, ORAC values rangedfrom 7.8 to 33.7 μmole TE/g of fresh berries and the ORAC values of theleaves range from 69.7 to 182.2 μmole TE/g (Wang S Y, Lin H S., J AgricFood Chem. 2000 February; 48(2): 140-6.). Comparatively, a fruit with anORAC value of 8 μmol TE/g of fresh berries will have an ORAC value ofabout 35 μmol TE/g of dried berries and an ORAC of 34 μmol TE/g of freshberries corresponds with an ORAC of 162 μmol TE/g dried matter. Blackraspberries have a very high ORAC of 77 μmole TE/g while boysenberrieshave an ORAC of 48 μmole TE/g, and red raspberries and blueberries havean ORAC of 24 and 23 μmole TE/g, respectively.(http://www.deckerfarm.com/antioxidants.html). Acai is another fruithaving a high ORAC content (167 ORAC units/g according to BrunswickLaboratories). Other fruit having high ORAC values include pomegranate(33.1 μμμmole TE/g) and aronia berry (62 μμmole TE/g) as well as wholecoffee fruit extract (6250 ORAC units per gram according to BrunswickLaboratories). The stage at which the plant is harvested affects theORAC value. Blackberries have their highest ORAC values during the greenstages, whereas red raspberries have their highest ORAC values whenripe. The ORAC may be obtained by using the method described by the U.S.Department of Agriculture's Agricultural Research Service (Prior andCao, 83(4) J. AOAC INT. 950-6 (2000)). Additionally, testing for ORAC,H-ORAC, N-ORAC, S-ORAC, and ORAC-E (high throughput ORAC foroil-in-water emulsion) is available from Brunswick Laboratories(www.brunswicklabs.com).

Anthocyanins have been formulated in topical form. For example, U.S.Pat. Pub. 2004/0022818 provides a skin care composition comprising fruitparticles. Red and blue fruits, as well as other fruits which containanthocyanins are included in this composition. Similarly, U.S. Pub.2003/0161897 and U.S. Pat. No. 6,361,786 provide a topical antimicrobialfactor having fruit juice (cranberry, Aronia berry, blackberry, grape orblueberry) and polyvinylprolidinone. A topical oil-in-water emulsioncontaining a surfactant/emulsifier and a lipid, which may be ananthocyanin-containing berry extract, is provided by WO 98/05294. U.S.Pat. No. 5,011,855 provides a cosmetic composition having a-linolenicacid and an oil extracted from Ribes genus fruits.

However, there are problems associated with these and other uses ofantioxidants in a topical formulation. Particularly, antioxidants arereadily oxidized and lose their anti-oxidant capacity. Additionally,some of the oxidation products of antioxidants will have a deleteriouseffect; for example, vitamin C oxidizes to dehydroascorbate which isknown to lead to the formation of advanced glycation endproducts (AGEs)and therefore increase the signs of aging skin.

Therefore, a topical formulation containing an antioxidant with a muchhigher ORAC and consequently greater anti-oxidant power is needed incombination with a formulation that protects the antioxidant fromdegradation and loss of the anti-oxidative power to be used in treatingand protecting the skin.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a composition andmethod for treating and protecting the skin.

In accordance therewith, a method and composition is provided fortreating skin, comprising a topical composition containing macqui berryin a stabilized form and the administration of an effective amount ofthe topical composition to the skin.

In a preferred embodiment, the stabilized form of macqui berry includesa macqui berry or macqui berry extract and a glucuronide or glycuronide,or a dermatologically acceptable salt thereof.

In another embodiment, the stabilized form of macqui berry includes amacqui berry or macqui berry extract or a dermatologically acceptablesalt thereof, which has been encapsulated or placed in light-blockingpackaging.

Additional agents such as NADH, an antioxidant, an AGE inhibitor, acollagen enhancing agent, a mitochondrial resuscitant, a lightreflecting agent or a sunscreen, an anti-edemic agent, a glutathione oran inducer thereof, an anti-inflammatory agent, a phenylpropanoidglycoside, a depigmenting agent or agent addressing hyperpigmentation, askin-protective lipid, hyaluronic acid, an alpha hydroxy acid, an agentuseful for treating hormonal decline, an anti-acne agent, an agentaltering lipolytic activity, an anti-cellulitic agent, an agent alteringanti-capillary-fragility, an anti-elastase agent. an anti-erythemaagent; or an agent that raises cyclic AMP may additionally beincorporated into the macqui berry composition.

In one embodiment, in addition to the administration of topicalcomposition containing macqui berry in a stabilized form to the skin,macqui berry is also orally administered on its own or together withother antioxidants and synergistic compounds disclosed herein that aresuitable for oral administration. This increases the protective effecton the skin.

In yet another embodiment of the present invention, the stabilized formof macqui berry is used in a cosmetic. Since the macqui berryformulation is a natural dye as well as an antioxidant, the formulationprovides both color and antioxidation activity to the cosmetic.

Other features, advantages, and embodiments of the invention will beapparent to those skilled in the art from the following description andappended claims.

DETAILED DESCRIPTION

The present invention provides a formulation and method of use fortreating skin and reducing the signs of aging. This formulationcomprises macqui berry or an extract of macqui berry in a stabilizedformulation.

I. Macqui Berry

Macqui berry (Aristotelia Chilensis) comes from a berry plant indigenousto Chile and Argentina. The authocyanin content in the macqui berry ishigh. The juice has a total anthocyanin content of 203 mg anthocyaninper 100 ml juice. The ORAC of macqui berry 206 μmol TE/g (Cao, G, Prior,L. Agricultural Research, November 1996, p. 4-8 Brunswich Labs (2003)).This can be compared to other anthocyanin-containing fruit with high ormoderately high ORAC values, as shown in Table 1.

TABLE 1 ORAC value Fruit (μμmole TE/g) macqui berry 206 black raspberry77 aronia berry 62 boysenberry 48 pomegranate 33 red raspbererry 24blueberry 23

The macqui berry is particularly advantageous when used as a whole berryor partially purified extract. The berry or extract may be used as aliquid or juice, pulp, or in solid, dried form. Other compounds withinthe macqui berry are also advantageous for use in the topicalformulation of the present invention Macnuii berry contains a number ofanthocyanins including delfinidin-3-glucoarabinoside-5-glucoside;delfinidin 3,5-diglucoside-cyanidine-3-glucoarabinoside-5-glucoside;cyanidine-3,5-diglucoside; delfinidin-3-glucoarabinoside;delfinidin-5-glucoarabinoside; delfinidin-3-glucoside;cyaniding-3-glucoarabinoside; and cyaniding-3-glucoside.

The macqui berry used in the present invention may also contain only thefruit or the fruit in combination with other parts of the plant, such asthe leaves. Additional useful ingredients in the macqui includequercetin in the fruit and alkaloids in the leaves. These alkaloids aremostly indolic in nature and have anti-tumoral and antimicrobialactivities; they include maknmin, aristotelinone, aristoteline,aristotelone, aristotelinine, aristone, and others. Other agents foundin the fruit include n-noncoane, β-sitoesterol, antraquinone, andseveral triterpenes. The content of the macqui berry has been describedby “Patagonal® Natural Antioxidants” BDS Nutraceuticals and DKSH MarketIntelligence product brochure; Cesped, C. et al., Phytochemistry (1993)34(3) 881-2; Cesped, C., et al, Phytochemistry, (1990) 29(4)1354-5;Munoz, O. et al, (2001) Medicinal plants in Chile: Chemistry andPharmacognosis Editorial Universitaria University of Chile 330; andMiranda-Rottmnann. E., et al., (2002) J. Agricul. Food Chem. 50, 7542-7.However, these references do not provide a topical formulation that canbe used for the prevention and/or treatment of damaged skin.

Quercetin, found in the fruit of the macqui berry, is a phenolicantioxidant. The pharmacological actions of phenolic antioxidants areprimarily due to free radical scavenging and metal chelating properties.They also affect cell signaling pathways and gene expression. The ORACactivity order flavonol aglycones decreases in the order quercetin,myricetin and kaempferol (Soobrattee, M A, et al., Mutat Res. 2005 Aug.25). Quercetin has been used topically for the treatment of symptomaticdiabetic peripheral neuropathy due to oxidative stress (Valensi P, JDiabetes Complications. 2005 September-October; 19(5):247-53.)Therefore, the presence of this ingredient in the berry is beneficialfor the topical formulation.

Alkaloids, including makonin, aristotelinone, aristoteline,aristotelone, aristotelinine, aristone, have antimicrobial propertiesand therefore can be used in a topical preparation against acne vulgarisor to reduce the amount of preservatives needed in the topicalformulation. Their anti-tumor effect may prove beneficial in preventingdamage to skin from environmental tumor-inducing pollutants.

N-nonacosane is an anti-mutagenic. This is an important function inskincare as skin is exposed to pollutants and UV radiation on a dailybasis that enhance the occurrence of mutation in the cells. Therefore,the combination of the anthocyanins and n-nonasosane in the formulationof the present invention are particularly advantageous in a topicalformulation.

β-sitosterol is anti-inflammatory (Lawrence Review of Natural Products,1995) anti-bacterial (Internat. J. Crude Drug Res. 28(1,2,3,4): 1990,page 155), anti-androgenic (Malini, T. and Vanithakumari, G. 1989,Journal of Ethnopharmacology, 28: 221-234, 1990), and an antioxidant,making it a valuable ingredient for topical formulations for skin damagefrom aging, acne or rosacea.

Antraquinone, and it derivatives are known to have antibacterialproperties. (Chen C H, Yao Xue Xue Bao 1964; 11(4):258-65). Therefore,the combination of this agent with the anthocyanins is particularlyadvantageous due to the disinfectant properties of the formulation.

Therefore, the interactions between the different constituents of themacqui berry make it a novel, potent ingredient for topical applicationto address skin aging.

The macqui berry may be used as a whole fruit, a fruit juice, or anextract of the fruit with the optional addition of extract from theleaves or stem of the plant. The extract may be used in a liquid orsolid form.

II. Stabilizer

Since macqui berry contains antioxidants having a high ORAC ratio, it isrelatively unstable over time, especially when exposed to light and/orair. Therefore, the present invention provides a formulation where theantioxidant from the macqui berry is stabilized. Stabilizing agentsinclude, for example, glucuronide, glucuronide, microencapsulation, andlight- and/or air-blocking packaging. Other stabilizing agents such asdiethylhexyl syringylidene malonate may be used. In one embodiment ofthe present invention, two or more different stabilizers are be used.

Flavonoid Glucuronides and Glycuronides

Flavonoid glucuronides and flavonoid glycuronides are added to theformulation of the current invention as a stabilizer for the macquiberry. As used herein, the term “flavonoid glucuronide” encompassesflavonoids that are attached to a glucuronic acid (e.g., glucose havinga carboxylic acid at the C6 position on the sugar ring); the term alsoencompasses flavonoid glucosides, which are flavonoids attached toglucose. Similarly, flavonoid glycuronides are flavonoids attached toglycuronic acid, and flavonoid glycosides, which are herein encompassedin the term flavonoid glycuronides, are flavonoids attached to aglycose.

Exemplary flavonoid glucuronides include: luteolin 7-glucuronide;luteolin 3′-glucuronide; luteolin 7-diglucuronide; luteolin7-glucuronide-3′-ferulyglucaside; apigenin 7-glucuronide; quercetin3-(isoferulylglucuronide); 7-sulfatoglucuronides of tricin and luteolin;3-glucuramnide-7-sulfate of kaempferol, quercetin, or isorbamnetin;quercetin 3-glucuronide-3′-sulfate; gossypetin 8-glucuronide-3-sulfate;rhanmetin 3′-glucuronide-3,5,4′-trisulfate; the 7-glucuronide and8-glucuronide of 5,7,8-trihydroxyflavone (norwogonin),5,7,2′-trihydroxyflavone 7-glucuronide; apigenin7-rhamnosyl-(1->2)-galacturonide; apigenin 7-digalacturonide; apigenin7-galacturonyl glucoside; apigenin 7-sulfatoglucuromde;5,6,7,2′-tetrahydroxyflavone 7-glucuronide;5,7,2′-trihydroxy-8-methoxyflavone 7-glucuronide5,7-dihydroxy-8,2′-dimethoxyflavone 7-glucuronide; luteolin7-galacturonide-4′-glucoside; 8-hydroxyluteolin 4′-methyl ether8-glucuronide; tricetin 7,3′-diglucuronide; ticetin 3′-methyl ether7,5′-diglucuronide; aponetzgerin 7-glucuronide; 8 hydroxytricetin7-glucuronide; kaempferol 3-rhamaoside-7-galacturonide; kaenmpferol3-glucoside-7-glucuronide; eupafolin 3-glucuronide; herbacetin3-glucuronide-8-glucoside; quercetin 3-glucoside-7-glucuronide;quercetin 3-gentiobioside-7-glucuronide; quercetin3-glucuronide-3′-sulfate; tamarixetin 5-glucoside-7-glucuronide;quercetin 3′,4′-dimethyl ether 5-glucoside-7-glucuronide; gossypetin3-glucoside-8-glucuronide; and gossypetin 3-glucuronide-8-glucoside.

Exemplary flavonoid glycuronides include 7-glycuronide luteolin and7-glycuronide apigenin.

A particularly useful flavonoid glucuronide is the glucuronide derivedfrom rosemary. Rosmnarinic acid is a naturally-occurring flavonoidisolated from various plants, such as Rosmarinus officinalis (RicercaSci. 1958, 28, 2329), Melissa officinalis (Arch. Pharm. 1960, 293,1043), and Teucrium scorodonia (Planta Med. 1965, 13: 3, 331).Rosmarinic acid may be obtained from these and other plants byextraction, (see U.S. Pat. No. 5,908,650), and also may be obtained fromplant cell cultures, such as Coleus blumei (see Naturwissenschaften1977, 64:11, 585; Sec also Liu, G., et al., Biochem Pharmacol., 1992 43,147-152.)

Flavonoid glucuronides and glycuronides have been found to be useful instabilizing, (e.g., reducing the rate of loss of anthocyanin color,intensity, and aromaticity, due to pH, heat, and/or light)anthocyanin-containing foods and beverages. See U.S. Pat. No. 5,908,650.As well as protecting the color of the anthocyanin and creating a colorshift, flavonoid glucuronides and glycuronides can increase thestability of the macqui berry topical formulation, and allow forincreased antioxidant activity both in the initial formulation of thetopical composition and over time. This is particularly useful fortopical formulations where the compound will likely sit on a shelf for aperiod of time before use in a water-based gel, serum, or oil-in-wateremulsion.

Many of these glucuronides, such as rosmarinic acid, have antioxidantproperties as well as stabilization properties. Therefore, the use ofthese glucuronides is advantageous in the present invention because ofthe additional antioxidative effective on the skin and underlying tissueof the composition. Rosmarinic acid has an IC₅₀ of 0.74 μM.

The flavonoid glucuronide or glycuronide is incorporated in the presentcomposition in an amount effective to protect and stabilize theanthocyanin. This amount will depend on the amount and type ofanthocyanin in the formulation; therefore, the ratio of flavonoidglucuronide or glycuronide to the macqui berry anthocyanins will vary toachieve effective stabilization of the anthocyanins. It is contemplatedthat the weight of flavonoid glucuronide used will be between 0.1% and500% of the weight of anthocyanin used in the formulation. Preferably,the weight of flavonoid glucuronide or glycuronide is between about0.2%-10% of the weight of anthocyanins. In a more preferred embodiment,the weight of flavonoid glucuronide or glycuronide is between about0.5%-5% of the weight of anthocyanins.

In one embodiment of the present invention, the stabilizer is astabilizer such as those described in U.S. Pat. Pub. 2005/0244349,herein incorporated by reference. These compounds are taught to beuseful for stabilizing photosensitive poly-unsaturated and/or aromaticcompound such as antioxidants against degradation from sun light, heatand air oxidation. The stabilizers include dialkylbenzalmalonates,monoalkyl-monoacyl benzalmalonate, dialkyl benzalmalon-amide andmono-alkyl-monoacyl benzalmalonamide compounds, and are able tostabilize photo-sensitive ingredients within the macqui berryformulation of the present invention. In particular, a preferredstabilizer is diethylhexyl syringylidene malonate.

Encapsulation

In one aspect of the present invention, the macqui berry formulation isstabilized using encapsulation, or more preferably, microencapsulation.Microencapsulation can protect the anthocyanin from the oxidativeeffects of the surrounding environment and increase the effectiveness asan anti-aging agent. Processes conventionally used formicroencapsulation may be employed, and may comprise encapsulation bynanosomes, liposomes, or other vehicles known in the art.

The microcapsules may be prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.(Remington's Pharmaceutical Sciences, A. Osol ed., 16th ed. (1980)).Microencapsulation is particularly useful for formulations containingDHLA, which is prone to degradation and oxidation. In one preferredembodiment, the microencapsulating agent is biodegradable, such as acarbohydrate, a naturally occurring polymer, or lecithin.

One typical process is to dissolve the shell material in a solvent (inthe form of a colloidal or true solution) and to disperse the corematerial in the resulting solution in the form of solids ormicro-droplets. This dispersion is divided into micro-droplets and thenheated using, for example, hot air. During this process, the solventevaporates and the shell material re-precipitates in the form of solidsand forms a shell around the core material. This gives crudemicrocapsules, which can then be subjected to the customary processingsteps and incorporated into the final formulations. This processutilizes the known phenomenon of coacervation.

Another process of microencapsulation uses interface polymerization tocreate the microcapsule shell. In this method, precursors of the shellmaterial, for example monomers, are concentrated onto the core material,where they polymerize to give the final shell film. Fat-coatingprocesses also may be used.

The materials used for microencapsulation are selected from conventionalhydrophilic or hydrophobic substances or mixtures thereof. Solids, inparticular natural polymers, for example, starch and otherpolysaccharides, are preferred. However, synthetic polymers can also beused. Examples of shell materials are fats and/or waxes, preferablythose having a solidification temperature of approximately 35°-80° C.and include mixtures of cetyl palmitate and cetyl alcohol. Othercompounds include polysaccharides and their derivatives of natural orpartially synthetic origin, (e.g. cellulose derivatives); further,polymers of α- and/or β-hydroxycarboxylic acids, in particular polymersof glycolic acid (polyglycolides), lactic acid (polylactides),α-hydroxybutyric acid (polyhydroxybutyrate), α-hydroxyvaleric acid(polyhydroxyvalerate) and/or their copolymers, or mixtures of suchpolymers and/or copolymers.

Independently of the specific technique for preparing the microcapsules,it is preferred to carry out the process at a temperature which does notcause any of the components of the formulation to decompose or losetheir antioxidant activity.

Similarly, nanoencapsulation may be used. Nanoemulsions are meta-stableoil-in-water emulsions having a globule size is less than 150 nm. Theycan be stabilized with amphiphilic lipids. Nanoemulsions arestructurally distinct from microemulsions which are thermodynamicallystable dispersions comprising micelles of at least one amphiphilic lipidswollen with oil and do not require mechanical energy to be prepared. Anadvantage of using nanoencapsulation is the reduced need forsurfactants, which may tend to lead to intolerance and entailing asticky feel when applied to the skin (see U.S. Pat. No. 6,562,356).

In one embodiment, the formulation is encapsulated in cyclodextrin. Suchprocess is performed, for instance, by Wacker Fine Chemicals(www.wacker.com).

In another embodiment, the macqui berry formulation is encapsulated withNADH, R-DHLA, ATP., Glutathione, and SOD in Nano Spheres. Such processis perfor .ed, for instance, by Salvona Technologies. In anotherembodiment, biopolymer nanoemulsions from Ivrea-Pak Tech are used toeliminate undesirable residue (“ghosting”) commonly associated withporous particulate entrapment formulations.

Packaging Material

The macqui berry formulation may be stabilized by placing theformulation in a packaging that blocks the radiation that causesoxidation of the berry components. Preferably, this package will blocklight from 450-750 nm, the visible range, which is known to damage mostnutrients. The packaging material will preferably have less than 1%transmittance, or more particularly less than 0.1% transmittance withinthe range from 450-750 nm. The packaging should not contain polyvinylchloride-containing polymers that degrade organic molecules tocarcinogenic compounds. Dark violet packaging plastic or glass may beused in a particular embodiment (mironglass.com.) The packaging may beadditionally air-less and therefore prevent air from getting in contactwith the formulation once opened. Additionally the packaging may benitrogen-flushed to further protect the formulation.

III. Additional Agents

In addition to the macqui berry, the topical formulation in manypreferred embodiments of this invention contains at least one or moreadditional active ingredients. A non-limiting list of such ingredientsis included herein.

Collagen or Elastin Enhancing Agents

The collagen or elastin enhancing agent may be administered by applyingthe agent to the skin. Collagen enhancing agents such as those describedherein may also be added to the present formulation. This increasedcollagen synthesis is an important aspect of the present invention, asit provides additional benefit to the skin and reduces the effects ofaging. Agents having ‘collagenic activities’ include the anthocyanidins,ascorbic acid, asiatic acid (such as from centella asiatica), aucubin,proanthocyanidins, stabilized vitamin C, the amino acids 1-lysine,1-proline and their derivatives (e.g., dipalmitoyl-hydroxy-praline,hydroxyproline, homoproline, and natural raw materials containing thesesuch as apt (Ahnfeltia concinna) available from CIR)), and copperpeptides. Agents having ‘collagenase-inhibitor activities’ include theanthocyanidins, eicosapentacnoic acid, proanthocyanidins such as grapeseed proanthocyanins, procyanidins, bovine cartilage extracts, andglycosaminoglycans from shark. Agents having ‘collagen-sparingactivities’ include caffeic acid, chlorogenic acid, cichoric acid,cynarin, and echinacoside. Each of these may be used in addition to theATP enhancement of the present invention. Alternatively, collagen itselfmay be added to the formulation, such as in the form of collagenpeptides (e.g. active collagen polypeptide available from Shanghai UChemCo. LTD.) or in a form adapted for delivery to the skin so that thecollagen will penetrate into the skin (e.g., the form described in U.S.Pat. No. 6,759,056).

Other collagen inducing agents are growth factors, such as EGI, FOF,TGF, TGF-β, HGH, NGF, KGF, IGF, HGF natural sources containing growthfactors such as colostrums (Pepha® Nutrix from Centerchem), deer antlerpreparations and peptides designed to increase the production of any ofthese growth factors (e.g., Syn®-col from Centerchem-TGF-β, HericiumErinaceus and Idebenone-NGF), or the production of collagen itself(e.g., Matrixyl300, Dermaxyl, and Calmosensine each from Sederma),collagen peptides and synthetic collagen inducing peptides (e.g.,pal-kttks). Collagen stimulating agents are those such as TGF-beta,retinoic acid and retinol derivatives, botanical and other naturalextracts such as Vigna Aconitifolia seed extracts, g-ascorbate andg-hcl.

Glucosamine, glucoseamine sulfate, glucosamine HCl, glucosamineascorbate, chondroitin sulfate and other glucosamine salts andderivatives may be used in the formulation to induce collagen. Manganesegluconate, a common source of manganese, may also be included in theformulation. The enzyme MnSOD, a powerful antioxidant that removessuperoxide radicals, may also be used.

In one embodiment, carnosine is added to the formulation to stimulatecollagen formation. Carnosine is degraded by histidine and carnosinaseto form histamine and β-alanine and thereby stimulates the biosynthesisof nucleic acids and collagen (Nagai, K et al., Surgery 1986, 100(5);815-821). Carnosine is able to react with carbonyl groups on glycated oroxidized proteins (i.e., camosinylation) and inhibit the glycoxidisedproteins from cross-linking with normal macromolecules and causing thesigns of aging (Hipkiss A R, et al., Mech Ageing Dev 2001 Sep. 15;122(13):1431-45; Hobart et al. Life Sci. 75:1379-89). In one embodiment,a silicon, or an ortho silica acid described in U.S. Pat. No. 5,922,360,may be used. This ingredient may be used to boost collagen production.

Carnosine, the dipeptide β-alanyl-L-histidine, and its related compoundssuch as anserine (β-alanyl-1-methyl-L-histidine) and homocarnosine(γ-amino-butyryl-L-histidine) as well as the closely related compoundcarcinine(β-alanyl-histamine) are preferred antioxidants exhibitingstrong and specific antioxidant properties that may be added to theformulation of the present invention. Carnosine is present in millimolarconcentrations in tissues, including skeletal muscle and brain, and isan anti-glycation agent and a free radical scavenger as well as anantioxidant. Useful carnosine include the dipeptideβ-alanyl-L-histidine, D,L-carnosine, D-carnosine, L-carnosine, thederivatives anserine and homocarnosine, as well as their salts, such aszinc carnosine, copper carnosine, and copper anserine (Hipkiss A R,Chana H, Biochem Biophys Res Commun. 248(1):28-32, 1998; Hipkiss A R etal., Ann NY Acad Sci, 854:37-53, 1998).

When carnosine is chelated to zinc or copper ions, the presence of theions enhances carnosine activity as a superoxide radical scavenger(Gulyaeva N.Y., Biochemistry 57 (7:2) 1051-4, 1987). Therefore, theaddition of carnosine as an agent in the present invention in chelatedform provides for superoxide scavenger activity as well as theanti-glycation and anti-oxidation properties of carnosine. Carnosine hasbeen administered at dosages above 500 mg/kg body weight in animalstudies and has been found to be safe at these levels.

Anti-Glycation Agents

Anti-glycation agents may be used in the present invention both toprotect the ATP enhancing agent and for the anti-glycation affect thatdecreases the signs of aging in the skin. The anti-glycation agent willprotect the ATP enhancing agent by removing free radicals that wouldinactivate the ATP enhancing agent or counteract the glycation effect ofthe ATP enhancing agent.

As used herein, the term “anti-glycation agent” means a compound usefulfor preventing and/or reducing the glycation of skin proteins, inparticular of dermal proteins such as collagen. The anti-glycation agentinhibits the formation of advanced glycation end products (AGEs) and isalso known as an AGE inhibitor.

Examples of anti-glycation agents are plant extracts of the Ericaceaefamily, such as an extract of blueberry (Vaccinium angustifolium),ergothioneine and its derivatives; and hydroxystilbenes and theirderivatives, such as resveratrol and 3,3′,5,5′-tetra-hydroxystilbene.Other exemplary inhibitors of AGE formation include, but are not limitedto, benfotiamine, pyridoxamine, G-rutin (Nagasawa T., Mol Cell Biochem.249(1-2):3-10, 2003); pyridoxal phosphate, aminoguanidine, aaminoguanidine-pyridoxal adduct, green tea (Ouyang P. Di Yi Jun Yi DaXue Xue Bao 24(3): 247-51, 2004); extracts of Thymus vulgaria (Morimitsuet al., Biosci Biotechnol Biochem 59(11):2018-21, 1995);Ge-132(2-carboxyethyl germanium sequioxide) (Unakar et al., Exp. EyeRes. 61(2): 155-64, 1995); curcumine (Sajithlal et al., BiochemPharmacol. 56(12):1607-14, 1998); extracts of Cratoxylum cochinchinense(Tang, S Y et al., Free Radic. Biol. Med. 36(12):1575-87, 2004);extracts of Apocynum venetum Luobuma (Yokozwa at et al., al., Food Chem.Toxicol. 42(6):975-81, 2004); carnosine; carnosinylated proteins(Hipkiss A R et al., Cell Mol Life Sci. 57(5):747-53, 2000); extracts ofEugenia bicyclis (Okada et al., Nat. Prod. 67(1):103-5, 2004); rutin(Kiho, T et al., Biosci. Biotechnol. Biochem. 68(1):200-5, 2004);amadoriase enzymes from Aspergillus fungi (Monnier V M at al., Biochem.Soc. Trans 31:1349-53, 2003; U.S. Pat. No. 6,605,642); guanidine richextracts of Galega officinalis; and extracts of lycoperrsiconesculentum. The AGE inhibitor as described herein may be incorporated inamounts from about 0.001%-30% by weight. More preferably, the AGEinhibitor is incorporated in amounts from about 0.1%-5% by weight, basedof the total weight of the preparation.

One preferred anti-glycation agent is carnosine. Carnosine, discussedabove for its collagen enhancing properties, is also useful as ananti-glycation agent. Carnosine also promotes wound healing (Roberts PR, et al., Nutrition 1998; 14; 266-9), protects against radiationdamage, is potentially a modulator of enzymatic activities, and has beenshown to be a chelator of heavy metals (Quinn, P J et al., Mol. AsptsMed. 1992; 13(5), 379-444; (Hipkiss A R. Int J Biochem Cell Biol 1998;30:863-8). Carnosine reacts strongly with aldehyde and keto groups ofsugars by Amadori reaction, and is also theorized to deplete certainglycolysis intermediates. Therefore, a reduction of glycolysisintermediates by carnosine depletes their energy supply. But theaddition of pyruvate reverses this effect. (Holliday R Br J Cancer. 1996April; 73(8):966-71). Therefore, it is preferable to add carnosine in aformulation with an agent which also reduces the amount of pyruvateavailable. Additionally, the reaction between carnosine and aldehydesprotects susceptible macromolecules. Therefore, carnosine inhibitsnonenzymic glycosylation and cross-linking of proteins induced byreactive aldehydes (aldose and ketose sugars, certain triose glycolyticintermediates and malondialdehyde (MDA), a lipid peroxidation product).(Hipkiss A R, et al., Ann N Y Acad Sci. 1998 Nov. 20; 854:37-53).

An anti-glycation agent of interest is garcinol. Garcinol occursnaturally in the latex exudate of the herb Garcinia Cambogia, which isused as a weight loss supplement. Garcinol is a moderate antioxidant,metal chelator, and free radical scavenger. It also is a superoxideanion scavenger and has been shown to suppress glycation in a bovineserum albumin/fructose system. (Yamaguchi F. et al., J Agric Food Chem,2000 February; 48(2): 180-5). It has also been shown that the(−)-hydroxycitrate from Garcinia fruits may aid endurance duringpost-absorptive aerobic exercise by promoting gluconeogenesis. Garciniais particularly useful as an additional agent because the combination ofgarcinol with carnitine and chromium will have anti-glycation propertiesand promote gluconeogneogenesis (McCarty M F. Med Hypotheses. 1995September; 45(3):247-54).

Aglycal LS 8777, made by Laboratoires Serobiologique (Cognis France),may also be included as an anti-glycation agent in the formulation ofthe present invention. Aglycal LS 8777 is a plant-based complex thatretards the glycation of proteins. This photo-complex aids in thelong-term elasticity of the skin and protects against the fragmentationof collagen (www.laboratoires-serobiologiques.com)

Aldenine, made by Lipotec (Spain), is a complex of a tripeptide andhydrolyzed wheat and soy proteins that boosts Collagen III synthesiswhile protecting cells from photo damage. Aldenine detoxifies the skinfrom harmful RCS (Reactive Carbonyl Species).

Another anti-glycation agent, ANTIGLYS_KIN from Silab, is rich inphenolic acids and glycopeptides from sunflower and inhibits the proteinglycation reaction and prevents the glyco-oxidation.

Compounds obtained from Pterocarpus marsupium may also be incorporatedinto the topical formulation. (−) Epicatechin, the active ingredient inthe Indian herb Pterocarpus marsupium Roxb, can be obtained from thewater extract of the bark and is insulinogenic. (Abroad F, et al., ActaDiabetol Lat. 1989 October-December; 26(4):291-300). It has been foundto decrease hepatic and skeletal muscle glycogen (Grover J K, et al.,Mol Cell Biochem. 2002 December; 241(1-2):53-9). In addition, threeflavonoid antioxidants are also present in the heartwood; theseflavonoid are marsupsin, pterosupin, and liquiritigenin. The gum tannicacid and a non-glucosidal tannin, kino tannic acid, and Pterocarpusmarsupium extracts have also been shown to have anti-oxidant activity(Katiyar S K, et al., Photochem Photobiol. 1995 November; 62(5):855-61)and a strong anti-glycation agent(www.laboratoires-serobiologiques.com/LSvi/english/prod.sub.-2.html)).

N-Acetylcysteine is an N-acetylated cysteine which is a thiol containingamino acid, also called α-acetamido-β-mercaptopropanoic acid, which is apreferred additional component of the present invention. Theincorporation of N-acetylcysteine into the topical formulation willimprove the signs of aging of the skin. N-acetylcysteine is anantioxidant and also has been indicated as protective against pulmonaryoxygen toxicity (Eur. Respir. 2: 116-126 (1989)). It is also ananti-glycation agent. Preferred forms of N-acetyl cysteine include:N-acetyl-L-cysteine, N-acetyl-L-cysteine amide, N-acetyl-L-cysteinemethyl ester, N-acetyl-L-cysteine ethyl ester, N-acetyl-L-cysteinepropyl ester, and N-acetyl-L-cysteine isopropyl ester. See PCTUS96/16534 which teaches topical compositions containingN-acetylcysteine, and U.S. Pat. Pub. 2003/0229141 which discloses thetopical use of N-acetyl cysteine to alleviate or improve variouscosmetic conditions and dermatological disorders.

One preferred AGE inhibitor is benfotiamine and benfotiamine derivativessuch as S-Benzoylthiamine O-monophosphate. Benfotiamine is the mostpotent of the allithiamines, a unique class of thiamine-derivedcompounds present in trace quantities in roasted crushed garlic andother vegetables from the Allium genus (such as onions, shallots, andleeks). Benfotiamine's unique open-ringed structure makes it able topass directly through cell membranes, readily crossing the intestinalwall and being taken straight into the cell, and is absorbed by the bodybetter than thiamine itself, and levels of thiamine and thiaminepyrophosphate remain higher for longer, thereby decreasing the formationof AGEs.

Another preferred anti-glycation agent is pyridoxamine. Pyridoxamine(4-aminomethyl-5-hydroxy-6-methyl-3-pyridinemethanol), and derivativesof pyridoxamine such as4-aminomethyl-5-hydroxy-6-methyl-3-pyridinemethanol dihydrochloride and4-aminomethyl-5-hydroxy-6-methyl-3-pyridylmethyl phosphate, may beincorporated in the ATP enhancing formulation of the present invention.Pyridoxamine is a vitamin B6 derivative which is water-soluble andnontoxic in rats and humans. It inhibits the formation of AGEs fromAmadori proteins and is classified as a post-Amadori inhibitor (Khalifahet al. Biochem. Biophys. Res. Comm. 199:257, p. 251-258). It is alsobelieved that pyridoxamine traps reactive dicarbonyl intermediates inAGE formation and may also decrease oxidative stress, which subsequentlydecreases AGE formation from reactive oxygen species (lacovella et al.SCJMM, 2004: 5, p. 73-101). Pyridoxamine has also been shown to inhibitadvanced lipoxidation end products (ALES) (Onorato J M. et al., J. Biol.Chem. 275(28):21177-84 (2000)). Decreasing ALEs formation isaccomplished by decreasing the concentration of an oxidiable substratesuch as glucose and blood lipids (Metz T O, et al., Arch BiochemBiophys. 419(1):41-9 (2003)). It has been proposed that the antioxidantproperties of pyridoxamnine be used for the inhibition of ALE as well asAGE formation and development of complications of diabetes andhyperlipidemia (Mene P, et al., Am J Cardiovase Drugs. 3(5):315-20(2003)). U.S. Pat. Nos. 6,750,209 and 6,740,668 demonstrate thedifference in pyridoxamine and the other 3B6 vitamins as inhibitors ofpost-Amadori antigenic AGE formation. The efficacy of inhibition ofoverall glycation of protein, in the presence of high concentrations ofsugar, was not predictive of the ability to inhibit the post-Amadoristeps of AGE formation where free sugar is removed. Pyridoxamine hasbeen shown to be the strongest AGE inhibitor of the B vitamins. (Price,D. L., J, Biol. Chem. 2001 Dec. 28; 276(52):48967-72).

The combination of benfotiamine and pyridoxamine for AGE inhibition isalso a preferred additional ingredient that may be added to theformulation of the present invention. This combination is discussed inU.S. Pat. Pub. 2006/0045896-A1, herein incorporated by reference

AGE inhibitors that may be added to the formulation include the enzymes,fructosyl lysine oxidase and fructose lysine 3-phosphosphokinase, whichcatalyze the deglycation reaction and generate free amine groups. Thebiochemical properties of these amadoriase enzymes and their role inprotein deglycation are described by Wu, X et al., Arch Biochem Biophys.419(1):16-24 (2003). See also Takahasi, M. et al., J. Biol. Chem. 272,3437-43 (1997). The amadoriase enzymes are particularly useful sincethey have strong anti-glycation activity, and some of these compoundsare selective for collagen. This makes these enzymes particularly usefulas components in the topical formulation of the present invention sincethey will preferentially act on collagen and therefore inhibit theglycation of collagen and reduce the signs of aging in the skin.

Another class of AGE inhibitors that may be used in the formulation ofthe present invention is described by Rahbar et al., Molecular CellBiology Research Commun. 3, 360-66 (2000). These compounds are benzoicacid derivatives, aryl and heterocyclic ureido compounds, and aryl andheterocyclic carboxamido phenoxy isobutyric acids. They have been shownto be potent inhibitors of glycation, and have been shown to inhibit theglycation of collagen. The compounds described by Wu, Takahasi, orRahbar may be used in combination with the formulation described herein.

Other AGE inhibitors may be added to the formulation of the presentinvention. Extracts of Paeonia suffruticosa have been shown to be AGEinhibitors (Okano et al., atwww.creative-developments.co.uk/papers/Natural %20Ingredients%201998.html)-. Additionally, AGE inhibitors have been isolated alongwith compounds having antioxidant activity from Paeonia suffruticosa;these compounds include the monoterpene glycoside,α-benzoyloxypaeoniflorin, β-benzoyloxypaeoniflorin, paeonolide,paeoniflorin and mudanpioside H. (Ryu G., et al, Arch Pharm Res. 2001April; 24(2):105-8). Another AGE inhibitor is from extracts ofSanguisorba officinalis, which has been shown to reduce chronicphotodamage to the skin. (Tsukahara K., Biol Pharm Bull. 2001 September;24(9):998-1003). Pterocarpus marsupium has been shown to be ananti-diabetic agent and strong antihyperglycemic agent (Babu P S., J.Pharm Pharmacol. 2004 November; 56(11):1435-42). C. Cochinchinense hasbeen found to be a particularly potent AGE inhibitor on proteins andalso to strongly inhibit hypochlorous acid-induced DNA damage. (Tang SY, Free Radio Biol Med. 2004 Jun. 15; 36(12):1575-87).

There are other natural products that are AGE inhibitors, which may beused in the present invention. The screening method described byMatsuura, based on a fluorometric analysis, may be used to determine theinhibitory index of the Maillard reaction and AGE inhibition todetermine compounds useful to include in the formulation of the presentinvention. (Nobuyasu Matsuura et al. J. Health Science 48(6) 520-526(2002)).

ATP-Enhancing or Encouraging Agents

Carnitine is a betaine that is an ATP-encouraging agent and is requiredfor the transport of long-chain fatty acids, ATP production, and removalof excess short- and medium-chain fatty acids. It is derived from theamino acid lysine. L-carnitine is the only biologically active isomer;however, the derivative acetyl-L-carnitine may also be used as the ATPenhancing agent. Acetyl-L-carnitine enters cells and crosses the bloodbrain barrier more effectively than L-carnitine (Kidd, P. M. 1999 Alt.Med. Rev. 4, 144-161).

Acetyl-L-carnitine has a three pronged anti-aging effect by being amitochondrial energy boosting agent, helping to boost acetyl-cholinenecessary for proper face muscle tone and being an effectiveantioxidant. It is useful in the transport of long-chain fatty acidsinto the mitochondrial matrix, transport of short- and medium-chainfatty acids away from the mitochondrial matrix, and regulation of energymetabolism through the modulation of acetyl CoA:CoA ratios. For thisregulation, the acetyl group of acetyl CoA is transferred to L-carnitineby carnitine acetyl-transferase (CAT), freeing CoA to participate in thePDH reaction. The acetyl-L-carnitine can then be removed from themitochondria (Arrigoni-Martelli E, et al., Drugs Exp Clin. Res. 2001;27(1):27-49; Rebouche C J. Carnitine. In: Shils M E, et al. eds.Nutrition in Health and Disease, 9th ed., Baltimore: Williams & Wilkins;1999:505-512). This increase of free CoA relative to acetyl CoA enhancesthe activity of pyruvate dehydrogenase (PDH) which catalyzes theconversion of pyruvate to acetyl CoA, a crucial reaction in glucosemetabolism(Ipi.oregonstate.edu/infocenter/othernuts/carnitine/carnitinerefs.html#re-f2).Propionyl-L-carnitine, which is not available in the US as an oralsupplement, cleaves into L-carnitine and propionate, which is useful asan intermediate during energy metabolism (Brass E. P., et al., J. Am.Coll. Nutr. 1998; 17(3):207-215). Useful carnitines include L-carnitine,acetyl-L-carnitine, propionyl-L-carnitine, acetyl-L-carnitine arginateand dermatologically acceptable salts thereof (e.g., acetyl-L-carnitinehydrochloride and acetyl-L-carnitine arginate dihydrochloride). Whilemeats, fish and dairy provide the richest sources of L-carnitine, it canalso be found in tempeh (fermented soybeans), wheat, (0.1 mg/slice ofbread), asparagus (0.4 mg/cup), and avocados (2 mg/ea). Generally, oralL-carnitine and acetyl-L-carnitine are available in doses from 500 mg to2,000 mg/day (Hendler S S, Rorvik D R, eds. PDR for NutritionalSupplements. Montvale: Medical Economics Company, Inc; 2001), and thetopical carnitine may be provided in similar or greater doses.

Coenzyme Q₁₀ (CoQ10, ubiquinone) is an ATP-encouraging agent that isuseful in the present invention. CoQ10 is a naturally occurring compoundthat is a strong antioxidant and has therapeutic potential for a numberof disorders, including congestive heart failure, muscular distrophy,periodontal disease, correction of drug-induced deficiencies, and immunediseases (AIDS, allergies). The biological activity of CoQ10 is believedto be linked to its ability to act as an antioxidant and free radicalscavenger protecting the integrity of cell membranes and offsetting theinability of diseased cells to manufacture sufficient energy forcellular repair, by stimulating mitochondrial respiration and productionof ATP. It has been shown to be effective in both clinical and cosmeticapplications. The solubility and bioavailability of CoQ10 used in thepresent invention can be enhanced by the method described in U.S. Pat.No. 6,632,443. In one embodiment, 10-300 mg CoQ10 is added to theformulation. In one particular embodiment of the invention, CoQ10 isall-trans CoQ10, as this form occurs naturally in human metabolism. Thiscan minimize the side effects and risks seen with the cis-form orcis-trans mixture. Trans-CoQ10 is available from Kaneka Corp, Japan asKANEKAQ10. This material is used in the nutraceutical industry and isnovel in its use for topical application. One particular embodimentcombines delta tacotrienols with all-trans Coq 10 for synergy, as deltatocotrienol is known to increase the endogenous production of CoQ10.

Coenzyme A (CoA) is an important ATP enhancing agent. CoA is a carrierof acetyl and acyl groups and is essential for numerous biosynthetic,energy-yielding, and degradative metabolic pathways. CoA is associatedwith the first step of the Krebs cycle, in which an acetyl group isintroduced into the cycle. Acetyl-CoA is the common cellular currencyfor acetyl transfers and has been used in the nutraceutical industry.CoA is derived from adenine, ribose, and pantothenic acid. The CoAprecursor, panthethine, which is a vitamin of the B complex, may also beused as an ATP-enhancing agent of the present invention. Panthethine ismanufactured by Daichi Fine Chemicals, Inc. as Pantesin LQ80.

Myocontracting Agents

In some embodiments, a myocontracting agent may be combined with themacqui berry. Myocontracting agents include, but are not limited to,choline and acetylcholinesterase inhibitors. Some particular agentsinclude huperzine, cerberin, eburnamonine, guanidion, histamine,leonurine, monocrotaline, panaxin, plumbagin, serotionin, solanine,solasonine, and theobromine.

Anti-Edema Ingredient

Edema is defined as soft tissue swelling due to expansion of theinterstitial volume. Agents that reduce edema are also useful as agentsin the topical formulation of the present invention. Proanthocyanidinswhich affect blood vessels have been reported in double-blind researchto reduce the duration of edema after face-lift surgery (Baroch et a.Ann Chir Polast Esthet 29:393-5 (1984)).

Lipolytic Ingredients

Lipolytic agents may also be applied as part of the topical formulationof the present invention. Theophyline is an agent useful as a lipolyticagent; it acts as both a localize diuretic and a lipolytic agent.Quinolinic acid, a structural analogue of nicotinic acid, inhibitsphosphodiesterase activity in adipocytes to increase AMP concentrationand increase lipolytic intensity. Other lipolytic ingredients useful inthe topical formulation of the present invention include cayenne, ColeusForskohlii, banaba extract, gugulsterone E & Z, bioprene, quinolinicacid, 3-n-butyl-phthalide, adenosine, ajoene, alginates, allicin, allin,amellin, bergapten, β-ecdysone, bromelain, chebulinic-acid, crocetin,cynarin, diallyl-disulfide, diallyl-suifide, diallyl-trisulfide,dipropyl-disulfide, forskolin, ginsenoside-rb-2, imperatorin, inulin,nicotinic-acid, ope, opcs, oxypeucedanin, phellopterin, polydatin,resveratrol, s-allyl-cysteine-sulfoxide, s-methyl-1-cysteine-sulfoxide,saikosaponin, wogonin, and xanthotoxin.

Cholinesterase Inhibitors and Acetyl-Cholinesterase Inhibitors

Cholinesterase (ChE) and acetyl-cholinesterase inhibitors (AChE) arepartially useful as a component in the topical formulation of thepresent invention because of their ability to augment the restoring of ayouthful tone to the skin.

The ChE inhibitor is preferably obtained from a plant source. PreferredChE inhibitors include, for example: (+)-carvone, demissine, 1-carvone,solanidine, 1,8-cineole, ephedrine, limonene-oxide, solanine,actinidine, eseramine, lycorine, solasodine, allicin, eseridine,palmatine, thymol, α-chaconine, fenchone, physostigmine, vasicinol,β-2-chaconine, galanthamine, pulegone, bufotenine, huperzine-a,sanguinarine, d-carvone, huperzine-b, selagine, demissidine, ibogaine,and serotonin.

AChE inhibitors are preferrably obtained from a plant source. Somepreferred AChE inhibitors are: (+)-menthol, berberastine, huperzine-a,menthone, (+)-piperitenone-oxide, berberine, isomenthol, p-cymene,(+)-pulegone, carvone, isomenthone, piperitenone, 1,8-cineole,chelerythrine, isopulegol, pulegone, akuammicine, d-carvone, 1-carvone,sanguinarine, akuammidine, d-limonene, i-limonene, terpinen-4-ol,alpha-terpinene, elemol, 1-menthol, viridiflorol, (+)-menthol,galanthamine, limonene, yohimbine, (+)-piperitenone-oxide,gamma-terpinene, menthol, menthone,

Preferably, the cholinergic substance is obtained from a plant source.Some preferred cholinergic substances are: arecoline, choline,deoxypeganine, deoxyvasicinone, eseridine, galanthamine, iridin,irigenin, lecithin, lithium, nicotine, nobiletin, physostigmnine,pilocarpine, pronuciferine, and yohimbine. Huperzine, an alkaloidderived from the herb Huperzia Serrata, is a preferred inhibitor ofacetylecholinesterase (AchE) used in the present invention. Huperzine isuseful in the forms huperzine A, huperzine B, 6-β-hydroxy huperzine A,and tautomers thereof. Transdermal application of huperzine has beenshown to improve memory and cognitive functions by adding huperzine witha permeation enhancer to increase blood plasma levels of huperzine.(U.S. Pat. Pub. 2004/020705). Similarly, huperzine can be administeredtopically for the treatment of Alzheimer's disease (U.S. Pat. No.6,352,715) and cholinergic deficient disorders (WO 2004 080436). In apreferred embodiment, the huperzine-a in the topical formulation isencapsulated in a liposome or nanosome.

Cytidine 5′-diphosphocoline, also known as citicoline or CDP-choline, isa preferred AchE inhibitor. Citicoline, or a stabilized form thereof(see U.S. Pat. Nos. 3,687,932 and 6,057,301), may be used.

Galanthamine is a reversibly acting cholinesterase inhibitor and anacetyleholinesterase inhibitor; it is a tetracyclic alkaloid which wasinitially isolated from galanthus nivalis. Galanthamine has uniquespecific properties, for example, highly analgesic effects comparable tothose of morphine, and is not as toxic as cholinesterase inhibitors suchas physostigmine and neostigmine. The principal use in humans has beenthe postoperative reversal of neuromuscular blockade. It has also beenadministered in a number of neuromuscular diseases, and has been shownto enhance activation of motor nerve terminals stimulated electrically,to increase ganglionic depolarization induced by acetylcholine, and toprotect against hexamethonium, indicating enhancement of the activity ofnicotinic receptors (U.S. Pat. No. 6,670,356). Galanthamine may beisolated, for example, by the process described in U.S. Pat. Nos.6,617,452 and 6,573,376, from either biological or synthetic material.

Glutathione

Glutathione, reduced glutathione, glutathione peroxidase, glutathiones-transferase or glutathione reductase may be incorporated in theformulation of the present invention. Additionally, synergisticintracellular glutathione inducers and precursors of glutathione may beused. These include omithine, α-ketoglutarate, 1-cystein, 1-glycine,1-glutamatic acid, glycyl-1-glytamine, n-acetyl-cystein, riboflavin,vigtamin B6, parsley seed or seed extract, sylimarin, and cysteine wheypeptides. Other ingredients synergistic with glutathione or glutathioneprecursors which may be added to the formulation include selenium salts,amino acid chelates (including methyl-1-selenocysteine,I-selenomethionine) and sacharomyces selenium ferment,polyenylphosphatidylcholine, myristicin (which may be isolated fromparsley), dihydromyristicin, quercetin, riboflavin, purselane extract,spinach extract, N-acetyl-cysteine,n-acetyl-glutammine,n,ndimethylglycine, anthocyanins, pycnogenol (pinus maritima) extract,grape seed extract, turmeric extract, sylimarin, tocopherols, r-lipoicacid, cynara scolymus extract, Picrorhiza kurrooa extract, Tinosporacordifolia extract, Phyllanthus niruri extract, Terminalia belericaextract, Terminalia chebula extract, Phyllanthus emblica extract,Boerhavia diffusa extract, Defensine (available from Silab), Phyllanthusamarus extract, Hibiscus rosa sinensis extract, gentisic acid, Tephrosiapurpurea, Andrographis paniculata extract, Occinum santum extract andcysteine peptides. Additionally, SUNACTYL® LS 9610 and AFR® LS 4467/VEG(from Laboratoires serobiologique) may be used.

Glutathione is a tripeptide consisting of the amino acids glutamic acid,cysteine, and glycine. Glutathione is important for the maintenance ofthe function of enzymes in cell metabolism. It assists in the transportof amino acids across cell membranes. It prevents oxidative alterationsof catalytic and allosteric centers, and upholds the optimumconformation of the enzymes for proper functioning (see WO 89/00427).The formation of cataracts is also associated with decreased levels ofglutathione. It has been suggested that NADPH production from D-glucoseaids in glutathione regeneration and protection from mitochondrialdysfunction thereby provide a neuroprotective effect. (Delgado-EstebanM, et al., J Neurochem. 2000 October; 75(4):1618-24). U.S. Pat. No.6,573,299 describes the addition of glutathione in a formulationcontaining a hydroxy acid. Glutathione has also been shown to be an ACEinhibitor (IC50=3.2 μg/ml), anticytotoxic, antieczemic, antihepatitic, acancer-preventive, and useful treating heavy metal poisoning. In thisformulation, glutathione is particularly useful as it is involved withpreventing both oxidative damage and detoxifying RCS (reactive carbonylspecies). When skin is exposed to UVB, endogenous is depleted and cannotperform its protective role. Therefore, replenishing can be a crucialstep towards preventing photoaging. Furthermore, detoxify RCS such as4-hydroxynonenal may form adducts with glutathiones and work with theformulation of the invention to reduce the signs of ageing. Thecombination of giutathione in a topical formulation therefore isparticularly useful.

Glutathione peroxidase catalyzes the reduction of hydroperoxides, suchas hydrogen peroxides, by reducing glutathione and protecting the cellfrom oxidative damage. Most glutathione peroxidase enzymes are tetramershaving four identical subunits containing selenocysteine in the activesite which participates directly in the two-electron reduction of theperoxide substrate. Glutathione is used as an electron donor toregenerate the reduced form of the selenocysteine (Forstrom, J. W., etal., Biochemistry 17, 2639-2644 (1978); Paglia, D. E., Valentine, W. N.J Lab Clin Med 70, 158-169 (1967). Glutathione peroxidase can be addedto a topical formulation to protect the skin for oxidative damage.

Glutathione also affects melanin in the skin. Melanin, or skin pigment,is produced by melanocytes. There are two common types of melanin inhair, eumelanin, which is brown/black in color, and phaeomelanin, whichis red/yellow in color. Glutathione has been implicated in thebiogenesis of the melanin precursor 5-S-cysteinyldopa and themelanogenic activity of pigment cells. Along with cysteine (which isused for glutathione biosynthesis), glutathione will effect the melaninand therefore coloration of the skin (Benathan M. et al., Cell Mol Biol(Noisy-le-grand). 1999 November; 45(7):981-90). Further,glutathione-reductase plays an important role in the regulation andcontrol of the biosynthetic activity of melanocytes. The differences inthe glutathione and the glutathione enzyme content in eumelanin andphaeomelanin pigmentation in skin indicate that the increase ofglutathione reductase activity in the environment of the melanocytes maystimulate the pigment cells to produce phaeomelanin instead of eumelaninpigment (Benedetto J P et al., J Invest Dermatol. 1982 December;79(6):422-4). Skin with no active mnelanocytes shows glutathione levelsintermediate between those of eumelanin and phaeomelanin. This isconsistent with glutathione reductase activity playing an important rolein the regulation and control of the biosynthetic activity ofmelanocytes. (Benedetto J P, et al., Invest Dermatol. 1981 November;77(5):402-5).

Glutathione also affects hyperpigmentation of the skin.Hyperpigmentation, caused by inflammatory skin disorders such as eczema,allergic contact dermatitis, and irritant contact dermatitis acne, isoften treated using sunscreen and an agent such as a hydroquinone,tretinoin, azelaic acid, or kojic acid (Pathak M A, Fitzpatrick T B,Nghiem P, Aghassi D S. Fitzpatrick T B, Editor. Dermatology in GeneralMedicine, 4th ed, New York: McGraw-Hill, pp 2742-60 (1993). Glutathioneplays a key role in the depigmenting and melanocytotoxic action of theseagents, which act to decrease intracellular glutathione by stimulatingpheomelanin rather than eumelanin, and thereby lighten hyperpigmentationof the skin. (Alena F, et al., Invest Dermatol 104(5):792-7 (1995)).Therefore, the use of the topical formulation of the current inventioncan be used to treat hyperpigmentation as well as affect the signs ofaging.

Antioxidants

In particular, preferred embodiments, in addition to the antioxidantspresent in the macqui berry, additional antioxidants may be added to thetopical formulation of the present invention. Exemplary antioxidantsinclude, but are not limited to, amino acids (e.g. glycine, histidine,tyrosine, and tryptophan) and their derivatives, imidazoles (e.g.urocanic acid) and their derivatives, carotenoids (e.g. lutein,lycopene), carotenes (e.g. α-carotene, β-carotene, lycopene,canthaxantin, cryptoxanthin, zeaxanthin and astaxanthin) and theirderivatives, chlorogenic acid and its derivatives, aurothioglucose,propylthiouracil, thiotaurine and other thiols (e.g. thioredoxin,cysteine, cystine, cystamine and their glycosyl, N-acetyl, methyl,ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl, γ-linoleyl,cholesteryl and glyceryl esters) and their salts, aminoethylcysteine,decarboxylated dimmer of amninoethylcysteine ketimine, dilaurylthiodipropionate, distearyl thiodipropionate, thiodipropionic acid andtheir derivatives (esters, ethers, peptides, lipids, nucleotides,nucleosides and salts) and sulphoximine compounds (e.g. buthioninesulphoximines, homocysteine sulphoximine, buthionine sulphones,pentathionine sulphoximine, hexathionine sulphoximine, heptathioninesulphoximine) in very low, acceptable doses (e.g. pmole to μmoles/kg);also (metal) chelating agents (e.g. α-hydroxy fatty, acids, palmiticacid, phytic acid, lactoferrin, tannins, and curcuumine), α-hydroxyacids (e.g. citric acid, lactic acid, malic acid, mandelic acid), humicacid, colic acid, colic extracts, bilirubin, biliverdin, EDTA, EGTA andtheir derivatives, unsaturated fatty acids and their derivatives (e.g.γ-linolenic acid, linolic acid, oleic acid), folic acid and theirderivatives, ubiquinone and ubiquinol and their derivatives, vitamin Aand derivatives (e.g. vitamin A palmitate), the B vitamins and theirderivatives, coniferyl benzoate of benzoin resin, rutinic acid and theirderivatives, butylhydroxy toluene, butylhydroxy anisole,nordihydroguaiacic acid, nordihydroguaiaretic acid,trihydroxybutyrophenone, uric acid and its derivatives, mannose and itsderivatives, sesamol, sesamolin, zinc and its derivatives (e.g. ZnO,ZnSO₄) including zinc amino acid chelates(zinc-methionine, zincacetyl-methionate), selenium and its derivatives (e.g. seleniummethionine), stilbenes and their derivatives (e.g. stilbene oxide,trans-stilbene oxide), proanthocyanidins, ascorbic acid, particularlyfat-soluble fatty acid esters of ascorbic acid (e.g. ascorbyl palrnitateand tetrahexydecyl ascorbate), quercetin and its derivatives (e.g.quercetin glycoside or rutin), hesperidine, sylimarin, sylibin,glabridin, superoxide dismutase and their derivatives, catalase and itsderivatives, carnmosic acid and its derivatives, apigenin and itsderivatives, luteolin and its derivatives, chiorogenic acid and itsderivatives, caffeic acid and its derivatives, ferrulic acid and itsderivatives, resveratrol and its derivatives, green tea polyphenols andits derivatives, matrix metalloproteinase inhibitors (e.g. green teapolyphenols, trans-retinoic acid, luteoline, quercetine, ursolic acid,shark cartilage preparations, diterpenes and ursolic acid fromSiegesbeckia and Centaurium extracts, and α tocopherol), Coenzyme Q10,glutathione and its derivatiatives, myristicin, changkil saponins (fromplatycodon grandiflorium, which is also known as jie geng),pomengranate, ellagic acid, honokiol (from magnolia officinalis),magnolol (from magnolia officinalis), naringenin, clove essential oil,martynosides, verbascosides, wolfberry (from lycium barbarum) extracts,cascading antioxidants including but not limited to carnosic acid,standardized extract of Phyllanthus emblica (trade named Emblica), pinusmaritima and pinus radiata bark extracts, hydroxytyrosol (from olives),genistein, thiotaurine, antioxidants from marine species (e.g.,Bioplasma® and monostrama extract from Seema), roxisomes (from AGI),crocetin, pine pollen extracts, beta glucans and the suitablederivatives of the invention (salts, esters, ethers, sugars,nucleotides, nucleosides, peptides and lipids) of these said activeingredients.

Anthocyanins in addition to the anthocyanins in the macque berry may beadded to the formulation of the present invention. These anthocyaninsmay be obtained from any portion of various plant sources, such as thefruit, flower, stem, leaves, root, bark, or seeds. One of skill in theart will understand that certain portions of the plant may containhigher natural levels of anthocyanins and, therefore, those portions arepreferably used to obtain the desired anthocyanins. Methods to determinewhether and which portions of a plant contain anthocyanins are known andnot discussed herein. The extraction and identification of variousanthocyanins are described, for example, in U.S. Pat. Nos. 6,818,234,6,780,442, and 4,413,004. Particularly preferred anthocyanins arederived from natural sources having high anthocyanin content.Approximately 300 anthocyanins have been discovered in nature, and comefrom sources including, but not limited to, acai berries, aroniaberries, apples, bilberries, black carrots, blueberries, cherries,cranberries, eggplants, elderberries, grapes, grapes, purple carrots,purple loosestrife, purple rice, radishes, raspberries, red cabbage,redcurrants, red-fleshed potatoes, red raspberries, red onions, speciesfrom the Rubus class (e.g, black raspberry, blackberry, and youngberry),species from the Ribus class (e.g., black currant and gooseberry), seabuckthorn, wolfberry extract, and strawberries.

Preferred anthocyanins that may be added to the formulation of thepresent invention include anthocyanis found in the extracts of red,blue, purple, magenta and black flowers. These flowers include, but arenot limited to purple dahlias, blue lotus, black tulips, and blackorchids. Therefore, in one preferred embodiment, the formulationincludes an extract of purple dahlia, an extract of black tulip, anextract of black orchid, or a combination thereof.

In one embodiment, an additional antioxidant included in theformulations of the present invention is one or more betacyanin.Betacyanins, like anthocyanins, may be obtained from natural sources andare antioxidants. One betacyanin of interest is betanin found in beets.

A lipoic acid is a preferred antioxidant for use in the topicalformulation of the present invention. Lipoic acid is available in boththe R and S forms. R-lipoic acid is a preferred form. Furthermore a formof R-Lipoic acid is preferred that does not readily polymerize such asR-lipoic nicotinate and certain sodium salt preparations of R-lipoicacid. The lipoic acid of the present invention also includes the reducedform, or dihydrolipoic acid. In aqueous systems, both lipoic acid andDHLA show strong antioxidant activity. Lipoic acid is also useful intreating diseases associated with oxidative stress including livercirrhosis, atheroschlerosis, and polyneuritis of diabetes mellitus.(Maitra, I., et al., Free Rad. Biol. Med. 18:823-829 (1995),introduction). The antioxidative activity of lipoic acid is due, atleast in part, to its ability to prevent free radical damage to cellsand cell components. Free radical damage is most evident in cellularmembranes because of the density of the molecular structure of themembranes. (R)-Lipoic acid has been shown to reverse the age-relateddecline in oxygen consumption and increase mitochondrial membranepotential. The age-related decline in hepatocllular glutathione andascorbic acid levels is reversed by treatment with (R)-lipoic acid (asan oral supplement in rats) (Hagen T M, et al., FASEB J. 1999 February;13(2):411-8).

Reduced R-lipoic acid, or R-dihydrolipoic acid (R-DHLA), may be usedinstead of or in addition to R-lipoic acid. R-DHLA which is formed insitu by the reduction of R-lipoic acid by NADH has more antioxidantproperties than lipoic acid. Both DHLA and lipoic acid havemetal-chelating capacity (LA chelates Fe²⁺ and Cu²⁺; DHLA chelates Cd²⁺)and can scavenge reactive oxygen species. However, only DELA canregenerate endogenous antioxidants and repair oxidative damage. DHLA canregenerate the endogenous antioxidants vitamin E, vitamin C andglutathione as well as provide peptide methionine sulfoxide reductasewith reducing equivalents. The reducing equivalents help in the repairof oxidatively damaged proteins such as a-i antiprotease (Biewenga G P.,et al., Gen Pharmnacol. 1997 September; 29(3):315-31). DHLA is a potentsulfhydryl reductant and has also been shown to act as a strong directchain-breaking antioxidant which may enhance the antioxidant potency ofother antioxidants such as ascorbate and vitamin E (Kagan V E, et al.,Biochem Pharmacol. 1992 Oct. 20; 44(8):1637-49).

Retinol and its derivates such as retinyl palmitate and trans-retinoicacid as well as retinols stabilized in liposomes and cyclodextrinpreparations may be added to the present invention. One retinol derivateof interest is tocopheryl-retinoate available from Nikko Chemicals Co.LTD.

Other agents useful in the topical formulation of the present inventioninclude vitamin C and the vitamin C derivatives including ascorbic acid,sodium ascorbate, and the fat soluble esters tetrahexyldecyl ascorbateand ascorbyl palmitate, magnesium ascorbyl phosphate,ascorbyl-glucoside, glucosamine ascorbate, ascorbyl acetate, etc.Additionally, extracts from plants containing a high amount of vitaminC, such as camu berry (Myrciaria dubia), accrola, emblica officinalis,and bioflavonoids from rose hip and citrus, may be used includingwatersoluble bioflavonoids such as hesperidin methyl chalcone.

In one particularly preferred embodiment, the antioxidant is superoxidedismutase (SOD) (and derivatives), catalase (and derivatives), or amixture thereof. In a particularly advantageous embodiment, SOD isheterologous SOD (HSDs), described in U.S. Pat. No. 6,426,068, which nolonger, or practically no longer, exhibit dismutase activity, but whichhave conserved their immuno-redox activity, stimulate the production ofendogenous SOD, as well as the production of catalase and of glutathioneperoxidase. In another embodiment, the macqui berry formulation containsa SOD and a lipid or protein such as ceramides, prolamines or polymerfilms based on prolamines (see U.S. Pat. No. 6,045,809). According toanother advantageous embodiment of said use, said plant heterologous SODis in particular derived from melon. The preferred embodiment uses anencapsulated SOD.

Sesame (Sesamum indicum) or sesame lignan may also be added to thepresent invention. Sesame and its lignans (the fibrous compoundsassociated with the sesame) act as antioxidants, reduce inflammation,normalize blood pressure, improve lipid levels, and promote fat burning.Sesame has also been shown to aid in the oxidation power orbioavailability of fish oil and conjugated linoleic acid, to enhance theanti-inflammatory effects of essential fatty acids, lower totalcholesterol and low-density lipoprotein (LDL), block oxidative damageimplicated in atherosclerosis, and reduce blood pressure. Sesame lignanscan dramatically increase tissue and serum levels of the vitamin Efractions α tocopherol and γ tocopherol, thereby enhancing theirprotective properties. (Yamashita K, et al., J Nutr. 1992;122(12):2440-6). Studies have shown that sesame can also reduceinflammatory processes known to promote cancer, senescence, and aging.

Sesame seed lignans significantly enhance vitamin E activity andincrease α tocopherol concentrations in the blood and tissue of rats feda diet containing α tocopherol and sesame seed or its lignans(Yamnashita K, et al., Lipids. 1995 December; 30(11):1019-28).Additionally, they elevate gamma tocopherol concentration by inhibitingan enzyme involved in breaking down tocopherols and tocotrienols (IkedaS, et al., J Nutr. 2002 June; 132(5):961-6).

Other preferred antioxidants which may be incorporated in theformulations of the present invention include tocopherols (e.g.d-α-tocopherol, d-β-tocopherol, d-γ-tocopherol, d-δ-tocopherol),tocotrienols (e.g. d-α-tocotrienol, d-β-tocotrienol, d-γ-tocotrienol,d-δ-tocotrienol) and vitamin E (α-tocopherol acetate). These compoundsadded to the compounds of the present invention may be isolated fromnatural sources, prepared by synthetic means, or mixtures thereof.Tocotrienol-enriched vitamin E preparations may be obtained byfractionating vitamin E preparations to remove a portion of tocopherolsand recover a preparation more highly concentrated in tocotrienol.Useful tocotrienols are natural products isolated from, for example,wheat germ oil, grain, or palm oil using high performance liquidchromatography, or isolated by alcohol extraction and/or moleculardistillation from barley, brewer's grain or oats. As used herein, theterm “tocotrienols” includes tocotrienol-rich-fractions obtained fromthese natural products as well as the pure compounds. The increasedglutathione peroxidase activity protects the skin from oxidative damage(Musalmah M, et al., Asia Pac J Clin Nutr. 2002; 11 Suppl 7:S448-51).

Gamma tocopherol is one particularly advantageous E vitamin since it iscapable of quenching reactive nitrogen oxide species such asperoxynitrite and nitrogen dioxide (Boje K M. Front Biosci. 2004 Feb. 1;9:763-76). Gamma tocopherol and its water-soluble metabolite,gamma-CEHC, have been shown to reduce inflammation by inhibitingprostaglandin E2 (Jiang Q, et al., Proc Natl. Acad Sci USA. 2000 Nov.10; 97(21): 11494-9) and gamma tocopherol administration correlates witha reduced risk from heart disease. (Kushi L H, et al. N Engl J Med. 1996Jun. 2; 334(18): 1156-62).

When α tocopherol or tocotrienol is added to the formulation of thepresent invention, it is also preferable to add sesame oil (or anextract thereof such as sesaminol, a sesame lignans) due to the enhancedantioxidant effect of the combination (Ghafoorunissa, Hemalatha S., etal., Mol Cell Biochem. 2004 July; 262(1-2): 195-202; Yamashita K, etal., Lipids. 2002 April; 37(4):351-8). One preferred formulationcontains d-α-, d-β-, d-γ-, and d-δ-tocopherol, d-α-, d-β-, d-γ-, andd-δ-tocotrienol in addition to the sesame lignans. Gamma-tocopherol,followed by δ and α tocopherol, has the highest content, with a reducedrisk from heart disease. (Kushi L H, et al. N Engl J Med 1996 Jun. 2;334(18):1156-62).

Vitamin A is a preferred addition to formulations of the presentinvention because of the increased stability it can impart to lipoicacids (Segall, A., J Cosmet Sci. 2004 September-October; 55(5):449-61).

In addition, carotenoids, particularly the xanthophyll type, are alsopreferred antioxidants that can be used in the practice of the instantinvention. The xanthopyll type carotenoids include molecules such aslutein, canthaxantin, cryptoxanthin, zeaxanthin and astaxanthin.Xanthophylls protect compounds such as vitamin A, vitamin E and othercarotenoids (Demmig-Adamas, B. Biochemica et Biophsyica Acta, 1020:1-24(1990)). Xanthophylls can be obtained from a multitude of naturalsources, or produced as described, for example, in U.S. Pat. No.5,916,791.

Flavan-3-ols are also preferred antioxidants that may be used in theformulations of the present invention; they belong to a class ofnutrients known as the flavonoid family. Particularly preferredflavan-3-ols include the procyanidin mixtures extracted from grape(Vitis vinifera) seed. Proanthocyanidins play a role in thestabilization of collagen and maintenance of elastin, two criticalproteins in connective tissue that support organs, joints, bloodvessels, and muscle (Mitcheva et a. Cell Mol Bio 39:443-8 (1993); Maffeiet al. Arzneimittelforsehung; 44:592-601 (1994)). Other flavan-3-ols mayalso be added to the formulation. These include catechin andepicatechin. Procyanidins are the dimers and oligomers of catechin andepicatechin and their gallic acid esters, and are widely distributed inthe plant kingdom. Other flavonoids, such as isoflavin P, quercetin,glabridin, red clover, and others described in U.S. Pat. Nos. 5,686,082and 5,686,367 may also be included in the formulation.

Myristicin. or 3-methoxy,4,5-methylendioxy-allylbenzene, is a non-amineprecursor of 3-methoxy-4,5-methylenedioxyamphetamine and may beincorporated in the formulation of the present invention. It can befound in Myristica fragrens (Nutmeg) contains approximately 4.0%myristicin and in parsley (Petroselinum crispum, Petroselinum hartense,Petroselinum sativum), which also contains apiol. Parsley has beentraditionally used as a diuretic, for colic, indigestion, and intestinalgas. Myristicin has been shown to induce production of a detoxifying,antioxidant enzyme called glutathione S-transferase, in mice (Ahmad H,et al., Biochem Biophys Res Commun. 1997 Jul. 30; 236(3):825-8).Myristicin can cause convulsions and nausea, and is toxic andhallucinogenic if taken in large amounts. However, smaller doses ofmyristicin have antioxidantive properties. An oral psychotropic dose ofmyristicin is 400 mg (see Stein U et al., Forensic Sci Int. 2001 Apr.15; 118(1):87-90; Zheng G Q, et al., Carcinogenesis, 1992 October;13(10): 1921-3). Parsley, a source of myristicin, may be used in thetopical formulation. However, the amount of parsley extract must belimited due to the fact that parsley can cause phototoxic reaction ifthere is a simultaneous exposure to sunlight. The rash is caused by thepsoralen furocoumarin found in the oil extract of parsley, and ispresent in other plants as well (celery, fennel, parsnips, limes,lemons, and figs) (see Lagey, K, Burns 1995 November; 21(7):542-3;Gruenwald J. PDR for Herbal Medicine. 1st ed. Montvale, N J: MedicalEconomics; 1998:1023-1024; Smith D M. Practitioner. 1985; 229:673-675;Stransky L, Tsankov N. ContactDermatitis. 1980; 6:233-234). Therefore,an extract containing myristicin and not furocoumarin may be used toprovide the antioxidative and protective effect of the myristicin butwithout the phototoxic reaction when applied to the skin.

Ethylenediaminetetraacetate (EDTA) or other metal chelators arepreferred antioxidants that can be included in the formulation. Planttannins are also metal chelators that may also be included in theformulation of the present invention. The chelating agent forms acomplex with metal ions, inactivating them, and preventing them fromfrom affecting antioxidant activity. Other chelators, in addition tothose described above, include, but are not limited to, dihydroxyethylglycine, citric acid, tartaric acid, and mixtures thereof.

In one preferred embodiment, a metal chelators is added to theformulation which may or may not include an additional antioxidant. Themetal chelating agent is particularly advantageous in that it furtherstabilizes the color and antioxidant property of the macqui berry ormacqui berry extract and the optional additional antioxidant.

An additional antioxidant that may be used in the present invention is aphenylpropanoid glycoside. Martynoside, a particularly preferredphenylpropanoid glycoside, may be isolated from a number of botanicalsources such as: Clerodendron trichotomum(apps1.niaid.nih.gov/struct_search); the aerial section of Scutellariapontica (Ersoz, T, et al., Turkish J. Chem. “Phenolic Compounds fromScutellaria pontica”, which also provides the isolation of otherphenylalkyloid glycosides); transformed root cultures of Catalpa ovata(Halina Wysokinska J, et al., Free Radic Res. 2003 August;37(8):829-33); Pedicularis plicata (Liao, R. et al., PhytotherapyResearch 1999 13(7):621-623; which also provides the isolation ofverbascoside); Pedicularis (Wang et al., Sci China C Life Sci (1996)39(2):154-8; which also provides the isolation of the phenyipropanoidglycosides: echinacoside, verbascoside, leucosceptoside a, andpediculariosides a, m and n). Their antioxidant scavenging activitiesare similar to those of the o-dihydroxy group of phenylpropanoidglycosides (Wang et al., Biochem Pharmacol (1996) 51(5):687-91). Theaddition of martynoside or verbascoside as an ingredient in theformulation of the current invention is particularly advantageousbecause these phenylpropanoid glycosides have been shown to reducefatigue in muscle tissue. This allows for a relaxation and smoothing inthe overlying skin and reduces the signs of aging (Liao, R. et al.,Phytotherapy Research 1999 13(7):621-623).

An additional antioxidant that may be used in the present invention iscoffee berry. Coffee berry is the fruit from a coffee tree or shrub, andhas an ORAC of 6250 g mole TE/g. The coffee berry is rich inpolyphenols, and contains phenolic acids at a concentration of 97 mg/100g (Mattila, P. et al., J Agric Food Chem. 2006, 54(19):7193-9). In onepreferred embodiment, an extract of the whole berry may be added as aadditional antioxidant. In other embodiments, extracts containing onlypart of the coffee berry, with or without the seed, may be used.

Green coffee extracts may also be added to the formulation of thepresent invention.

Hydroxycinnamic acid derivatives (3-caffeoylquinic acid (3-CQA),4-caffeolyquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA),5-feruloylquinic acid (5-FQA), 3,4-dicaffecylquinic acid (3,4-diCQA),3,5-dicaffeoylquinic acid (3,5-diCQA), and 4,5-dicaffoylquinic acid(4,5-diCQA)) are found in coffee berry seeds (i.e., green coffee beans).The diCQA hydroxycinnamic acid derivatives have a strong free radicalscavenging activity while the other hydroxycinnamic acid derivates alsoshow superoxide anion radical scavenging activity (Iwai K, et al., JAgric Food Chem. 2004 Jul. 28; 52(15):4893-8). U.S. Pat. No. 5,972,409describes a method of obtaining soluble extract from green coffee. Thegreen coffee extract may be provided from either the whole fruit or theseed. In another embodiment, caffeic acid may be added to theformulation.

In a preferred embodiment, the antioxidant is an antioxidant endogenousto the human body. These antioxidants include, but are not limited tosuperoxide dimutates (SODs, such as Mn, Zn, or Cu SODs), glutathione andthe glutathione enzymes, NADH, lipoic acid and the salts and metabolitesthereof, NAC, thioredoxin, and the tocopherols.

The amount of antioxidant in the formulation (added in addition to theanthocyanin) is preferably from about 0.001 to about 80% by weight,preferably from about 0.1 to about 5% by weight, based on the totalweight of the preparation.

In one embodiment, the additional antioxidant in the formulation isadded in a particular ratio compared to the antioxidant in the macquiberry or macqui berry extract. The ratio is determine to optimize (i.e.,increase) the ORAC value of the formulation containing the two or moreantioxidants. This ratio is determined through testing the ORAC valuesof compositions having various ratios, and may be done by the methods asdisclosed by Brunswick laboratories (www.brunswicklabs.com). Assays suchas those described by D. Huang et al. in J. Agric. Food Chem. 2005, 53,1841-1856 and D. Huang et al. in J. Agric. Food Chem. 2002, 50,4437-4444 may be used to determine ORAC.

Matrix Metalloprocase Inhibitors

Matrix metalloprotease (MMP) inhibitors may also be added to theformulation of the present invention. Most MMPs are thiols orhydroxyamates. Many MMPs are neutral zinc-dependant endopeptidases thatselectively catalyze the hydrolysis of polypeptide bonds; they degradeand rebuild structural proteins in collagens and are required for thehealing of moist skin wounds (Agren M S. Arch Dermatol Res. 1999 291(1L):583-90). Increased concentrations of MMP 1 (collagenase 1,interstitial collagenase), 3, (stromelysin 1), 7 (matrilysin, pump), 9(gelatinase b, 92kd gelatinase), and 12 (macrophage metalloelastase)have been found in sun-exposed skin. Additionally, increased levels ofMMP-1 have been found in smokers (Lahmann C, et al., Lancet 2001,357:935-936) MMP levels are also known to rise in fibroblasts as afunction of age, and oxidant stress is believed to underlie changesassociated with both photoaging and natural aging (Varani J, et al., JInvest Dermatol. 2000 114(3):480-6). Therefore the addition ofinhibitors of MMPs, particularly MMP-1, 3, 7, 9, and 11 in the topicalformulation of the present invention, is contemplated. MMP inhibitorsinclude the tissue inhibitors of metalloproteinases (TIMPS) which arethe natural inhibitors of MMP activity (Gomez, D E. et al. (1997) Eur.J. Cell Biol. 74:111.), and include compounds such as ilomastat(www.chemicon.com/product).

Anti-Inflammatory Agents

The free radicals associated with aging skin will often also induceinflammation in the skin and lack of skin immunity. Thereforeanti-inflammatory agents including NSAIDS, COX-2 inhibitors (e.g.,nexrutine, ursolic acid, quercetin, curcumine, and evodia extract)(Kang, S. S., et al., Nat. Prod. Sci., 1999, 5(2): 65-69) can beincluded in the formulation of the present invention.

Mitochondrial Resuscitants

Mitochondrial resuscitants may also be added to the macqui berryformulation. Mitochondrial decay in aging is a major driving forcebehind the aging process (Ann N Y Acad Sci. 2004 June; 1019:406-11; ProcNatl Acad Sci USA. 1994 91:10771-8). The mitochondria are thepowerhouses of the cell responsible for producing all cellular energyand convert carbohydrates and fatty acids into ATP. ATP is necessary forthe production of proteins, which declines with aging (e.g., collagenand elastin).

Agents useful as mitochondrial resuscitants include, but are not limitedto, lipoic acid (Ames B., Ann. N.Y. Acad. Sci. 1033: 108-116 (2004),carnitine, COQ10, CoA, NADH, FADH, succinic acid, creatine, D-ribose,5-phosphoribosyl-1-pyrophosphate (PRPP), Sepitonic M3® (containingmagnesium aspartate, zinc gluconate, copper gluconate), pyruvate,phosphoglycolipids, gymnostemma pentaphyllum, and cytochrome C.Additionally, agents including phenylbutylnitrone (PBN) and otherspintraps, such as the nitrone or nitroso spin traps described in U.S.Pat. No. 5,723,502 (N-t-butyl-α-phenylnitrone,3,5-dibromo-4-nitrosobenzenesulfonic acid, 5,5-dimethyl-1-pyrrolineN-oxide, 2-methyl-2-nitrosopropane, nitrosodisulfonic acid,α-(4-pyridyl-1-oxide)-N-t-butylnitrone, 3,3,5,5-tetramethylpyrrolineN-oxide, and 2,4,6-tri-t-butylnitrosobenzene) as well as hydroxylaminessuch as N-tert-butyl hydroxylamine may be used. These agents areantioxidants as well as mitochonrial resuscitants.

D-ribose is a naturally occurring five-carbon sugar found in all livingcells, and is a preferred mitochondrial resuscitant. It is not anessential nutrient, since it can be made in the body from othersubstances, such as glucose. ATP (adenosine triphosphate) requiresD-ribose, as do nucleotides, nucleotide coenzymes, and RNA (ribonucleicacid). D-ribose, in the form of ribonucleoside diphosphates, isconverted to deoxyribonucleoside diphosphates, precursor molecules forDNA. D-ribose in RNA and D-deoxyribose in DNA. When D-ribose is added tothe formulation, it is particularly preferred to additionally include anAGE inhibitor to prevent the potential glycating effect of d-ribose.

R-lipoic acid is implicated in mitochondrial energy production andprotection from free radicals. It has been shown to maintain microsomalprotein thiols, protect against hemolysis, protect against neurologicaldisorders, and protect against ischemia/reperfusion injury. R, notS-lipoic acid is produced by the body and decreases in concentrationduring the aging process (Pick U., et al., Biochem Biophys Res Commun.1995 Jan. 17; 206(2):724-30). The formulation of the present inventionreplenishes this vital substance as well as provides protection to theskin cells. R-lipoic acid is incorporated in the formulation in onepreferred embodiment of the present invention.

Acetyl-L-carnitine has a three pronged anti-aging effect by being amitochondrial energy boosting agent, helping to boost acetyl-cholinenecessary for proper face muscle tone and being an effectiveantioxidant. It is useful in the transport of long-chain fatty acidsinto the mitochondrial matrix, transport of short- and medium-chainfatty acids away from the mitochondrial matrix, and regulation of energymetabolism through the modulation of acetyl CoA:CoA ratios. For thisregulation, the acetyl group of acetyl CoA is transferred to L-carnitineby carnitine acetyl-transferase (CAT), freeing CoA to participate in thePDH reaction. The acetyl-L-carnitine can then be removed from themitochondria (Arrigoni-Martelli E, et al., Drugs Exp Clin Res. 2001;27(1):27-49; Rebouche C J. Carnitine. In: Shils M E, et al. eds.Nutrition in Health and Disease. 9th ed. Baltimore: Williams & Wilkins;1999:505-512). This increase of free CoA relative to acetyl CoA,enhances the activity of pyruvate dehydrogenase (PDH) which catalyzesthe conversion of pyruvate to acetyl CoA, a crucial reaction in glucosemetabolism(Ipi.oregonstate.edu/infocenter/othemutscarnitine/carnitinerefs.html#re-f2).

The macqui berry formulation may be combined with a combination of alipoic acid, a carnitine, and a carnosine. The usefulness of acombination having these three ingredients is described in U.S. patentapplication Ser. No. 11/388,908, herein incorporated by reference.

ATP, adenosine 5′-monophosphate (AMP), and their degradation productsmay also be administered directly as agents in the topical formulationof the present invent invention. U.S. Pat. No. 5,227,371 teaches theoral or topical administration of AMP, ATP or their degradation productsadenosine and inorganic phosphate to increase ATP levels. ExtracellularATP has been shown to help regulate vascular tone (Burnstock, G. andKennedy, C Circul. Res. 1986, 58, 319-330), promote muscle contractions(Burnstock, G. Pharmacol. Rev, 1972, 24, 509-581), and arresting tumorgrowth (U.S. Pat. No. 5,049,372). When ATP or other agents susceptibleto degradation are used, a stabilized form of the agent is preferred.

Transforming growth factor-beta (tgf-beta), a potent fibrogenic cytokinehas been shown to decreases intracellular glutathione content, andincreases collagen I mRNA content and collagen protein production. Theeffect of glutathione depletion on tgf-beta-stimulated collagenproduction may be mediated by facilitating reactive oxygen speciessignaling. (Liu R M et al., Am J Physiol Lung Cell Mol Physiol. 2004286(1):1121-8.) It is therefore contemplated to additional add tgf-betato the formulation of the present invention.

Depigmenting Agents

Depigmenting agents may be added as an additional agent in the presentinvention. Depigmenting agents include tyrosinase inhibitors suchhydroquinone and its derivatives (e.g., hydroquinone monomethyl ether,hydroquinone monoethyl ether, arbutin); soy and derivatives thereof,retinoids such as retinol; Kojic acid and its derivatives (e.g., kojicdipalmnitate); transexamic acid; vitamins such as niacin, vitamin C andits derivatives; azelaic acid; phytic acid, licorice; mulberry extracts;extracts from rumex species such as rumex crispus extract; chamomileextracts; green tea extracts; lactic acid, pearl extract, Tricholomamatsutake extract, magnesium-asorbyl-phosphate, edelweiss extract, sedumacre extract, arbutine, ergothione, phyllantus emblica extract, α-MSHantagonists such as Undecylenoyl phenylalanine, germanium, and GAiBA andsongyi mushroom. Bowman-Birk Inhibitors are described in U.S. Pat. No.6,750,229 (e.g., inhibitors derived from the leguminosae, solanaceae,gramineae or cucurbitaceae family). Dermalight® and Clariskin3® fromSilab are also depigmenting agents that can be used. Kinetin (N6furfuryladenine) is a 6-(R-amino)purine cytokinin and is described inU.S. Pat. Nos. 5,602,139, 5,164,394, and 5,021,422. It has been shown tohave anti-aging effects on the skin of dogs as well as the depigmentingeffects without adverse effects (Kimura T, Doi K., Rejuvenation Res.2004 Spring; 7(1):32-9).

Skin-Protective Lipids

Skin-protective lipids, such as ceramides, cerebrosides, essential fattyacids and botanical or marine oils containing these, calophylluminophyllum squalene, squalane, botanical oils and butters such as (sheabutter, meadowsweet oil and coconut oil) phospatidylserine, spingolipidsand natural materials containing them such as Conyza Canadensis,phospholipids and sulfatet sterols available from Vincience may also beadded to protect the skin. A particular embodiment comprises a krill oilrich in Omega 3 PUFAs (EPA and DHA) as well as in antioxidants such asvitamin A, E or astaxanthin. This lipid is particularly suitable for usein the formulation of the present invention due to its multiprongedeffects including antioxidant action (ORAC value of 378), energyproduction function and anti-inflammatory action.

Nicotinamide-adenine-dinucleotide (NADH), in its reduced form, is acoenzyme form of vitamin B3 (niacinamide), which occurs in all livingcells including human cells. Similarly,nicotinamide-adenine-phosphate-dinucleotide (NADPH) is thephosphoralated form of NADH, and as used herein, NADPH is included inthe term “NADH.” NADH may be added to the formulations of the presentinvention.

NADH

NADH stimulates the production of ATP (adenosine triphosphate) duringthe regulation and release of stored energy. Higher levels of NADH inthe cell allow for the release of energy. Because of this, the oraladministration has been used to NADH treat fatigue, chronic fatiguesyndrome, and fibromyalgia. There is also an indication that NADH may beuseful in treating Alzheimer's disease (U.S. Pat. No. 5,444,053),treating Parkinson's disease (U.S. Pat. Nos. 4,970,200 and 5,019,561),depression, improving memory and concentration, and enuurance.

The topical administration of NADH is described in U.S. Pat. No.5,952,312, which demonstrates that NADH is adsorbed by the skin andstimulates enzymes essential in the production of energy. When NADH isincluded in the topical formulation, an additional stabilizer whichinhibits oxidation of NADH may also be added. The stabilizer is NaHCO₃,ascorbic acid, sodium ascorbate, tocopherols, tocopherol acetate,polyvinylpyrolidone, or a combination thereof (U.S. Pat. No. 5,952,312).

The addition of NADH is useful in the formulation of the presentinvention when lipoic acid or DHLA is incorporated in the formulationbecause it allows for the conversion of R-lipoic acid into the moreactive DHLA. As DHLA works to remove the signs of aging in the skin, itis oxidized to the less effective R-lipoic acid. NADH will then reducethe lipoic acid to form DHLA, which then continues to remove the signsof aging. In formulations of the present invention, the addition of NADHis also useful because DHLA will be converted to lipoic acid uponcontact with an oxidizing species in the skin; the NADH then effectivelyconverts the lipoic acid back to DHLA. In one preferred embodiment, thetopical formulation of the present invention contains NADH (or NADPH) ata concentration of 0.1%-10%, or more preferably 1%-5%.

Sunscreen Agents

In some embodiments, an additional active ingredients included in theformulations of the present invention are compounds having sunscreeningaction. Sunscreening agents include, but are not limited to aminobenzoicacid, avobenzone, dioxybenzone, homosalate, lisadimate, menthylanthranilate, octocrylene, octyl methoxycinnamate, octyl salicylate,oxybenzone, padimate o, phenylbenzimidazole, roxadimate, sulisobenzone,titanium dioxide, trolamine salicylate, zinc oxide, hammamelitannin, andcombinations thereof (seewvw.nlm.nih.gov/medlineplus/druginfo/uspdi/202782.html). In oneparticular embodiment, the sunscreen agent or agents are naturallyoccurring substances such as zinc oxide, coffee oil, leuco-melanin, datepalm fruit melanin, and galanga extract (available from symrise). Othersubstances that protect from uv damage that may be used include such assanguinaria extract krameria triandra root extract (15% neolignans)metallothionein, 1,25-dihydroxyvitamin d3, and thymidin dinucleotide. Apreferred sun-protective extract is a polypodium leucomotos extract.This compound may be incorporated in the topical formulation or,alternatively, it may be provided as an oral supplement in addition tothe formulation of the present invention for increased protection fromUV damage (Middlekamp-hup et al., J Am Acad Dermatol, 2004, 51(6)910-918). One preferred sunscreen agent is isoamyl-p-methoxycinnamate(from galanga, available from symrise, GMBY & Co.); this compoundprovides an spf of greater than 30 using only natural ingredients(botanicals, antioxidants, coffe oil and microfine zinc oxide).Additional sunscreen agents include allantoin, aloesin, apigenin,caffeic-acid, chlorogenic-acid, ellagic-acid, esculetin, esculin,ferulic-acid, fraxetin, fraxin, lawsone, p-aminobenzoic-acid, paba,procyanidins, rutin, silymarin, squalene, and umbelliferone.

Anti-Erythema Ingredient

By adding an anti-erythema ingredient to the formula, an additionaleffect caused by the damaging UV radiation besides free radicalformation, is addressed. The reduction in redness accomplished byapplying the formulation of the present invention is due to anincorporation of aesculin, colchicines, esculin, glycyrrhetinic-acid,ope, opes, procyanidin-a-2, procyanidins, rutin, or silyrnarin into theforrulation, Silymarin is a mixture of at least 4 closely relatedfiavonolignans, 60 to 70% of which is a mixture of 2 diastereomers ofsilybin. It has been shown to increase patient serum levels ofglutathione and glutathione peroxidase. It also works to increasesuperoxide dismutase (SOD) activity of lymphocytes and erythrocytes, aswell as the expression of SOD in lymphocytes. Silymarin has beenadministered orally at a dosage of 420 mg/day. (Wellington K. Jarvis B,BioDrugs. 2001; 15(7):465-89).

Agents for Hormonal Decline

Hormonal decline is known to occur with aging; therefore a class ofsubstances replenishing and regulating these is useful in combinationwith the macqui berry formulation of the present invention. Anon-exclusive list of agents useful for treating hormonal decline isestriol, 7-keto-dhea, dhea, estrone, estradiol, progesterone, pregnone,pregnenolone, melatonin, soy isoflavons, phytoestrogens (back cohosh,red clover, sage, etc.), chrysin, diosgenin, vitex extract,diindolmethane, pueraria mirifica (puresterol available frombio-botanica), β-sitosterol, β-stigmasterol, betulin and derivativesthereof, conyza canadensis essential oil, and maca extract standardizedto macamrides.

Anti-Acne Agents

Anti-acne agents may also be combined with the formulation of thepresent invention. Since both free radicals and inflammation arecofactors in acne, especially in adult skin, a combination with one ormore anti-acne ingredient may be used in the topical formulation of thepresent invention. A non-limiting list of useful anit-acne agentsincludes (−)-epigallocatechin, (−)-epigallocatechin-gallate, α-pinene,α-terpineol, anacardic-acid, azelaic-acid, baicalein, berberastine,berberine, β-carotene, camphor, caryophyllene, cryptotanshinone,δ-cadinene, γ-linolenic-acid, indole, linoleic-acid, nerolidol, pufa,pufas, pyridoxine, resorcinol, selenium, sulfur, terpinen-4-ol; thymol,tin, and zinc.

Anti-Cellulitic Agents

Compounds having anti-cellulitic activities may be included as anadditional ingredient in the formulation of the present invention. Thesecompounds include, but are not limited to, aesculin, bromelain, esculin,theobromine, and theophylline. Additional anti-cellulitic compoundsinclude caffeine, di-indolmethane, anti-estrogenic botanicals,terminalia arjuna, garcinia cambodgia, dihydromyricetin as found inMyriceline by Provital, as well as a citrus aurantium extract (see U.S.Pat. No. 6,224,873). This compound does not cross the blood/brainbarrier and has minimal impact on α 1,2 and β1,2 receptors, and istherefore safer then caffeine, and/or ephedra containing agents. In oneembodiment, no stimulants having a systemic effect if absorbed into thebloodstream are incorporated into the formulation.

Anti-Edemic Agents

Compounds having an anti-edemic activity are useful in combination withthe formulation of the present invention. The prevention of swelling andfluid retention in the skin present in both face and body can be aidedby combining an agent such as one of the following to the compound ofthe present invention. These compounds include, but are not limited to,(e)-4-(3′,4′-dimethoxyphenyl)-but-3-en-ol,10-acetoxy-8-hydroxy-9-isobutyloxy-6-methoxythymol, 13′,ii8-biapigenin,4-vinyl-guaiacol, 7-methoxycoumarin, acetyl-11-keto-β-boswellic-acid,actinidoles, aescin, α-amyrin, amentoflavone, anagyrine, anisodamine,anthocyanoside, arbutin, aristolochic-acid, aromaticin, artemetin,ascorbic-acid, asiaticoside, astringenin, aucubin, baicalein, baicalin,bavachinin, berberastine, berberine, β-aescin, β-amyrin,β-boswellic-acid, β-damascenone, β-escin, β-sitosterol, betulinic-acid,boehmerol-acetate, boldine, boswellic-acid, bradykininase, brazilin,bromelain, caffeic-acid, caryophyllene, caryophyllene-oxide, catechin,cis-spiroether, coniferyl-aldehyde, coumarin, crotaloburine,cryptolepine, curcumin, damascenine, digitoxin, diosmin, ephedrine,ephedroxane, escin, eugenol, eupahyssopin, faradiol, faradiol-monoester,friedelan-3-β-ol, gentianine, gentiopieroside, germacrone, ginkgetin,ginsenoside-r-o, glucose, glycyrrhetic-acid, glycyrrhetinic-acid,glycyrhizin, gnaphaliin, hederagenin, helenalin, humulone,isoferulic-acid, kawain, lanceolarin, lapachol, lupeol, madecassoside,maslinic-acid, matricine, matrine, oleanolic-acid, ope, opes,oxoushinsunine, paeonol, papain, papaverine, piperine,proanthocyanidins, procyanidin, procyanidins, pseudoephedrine,quercitrin, resveratrol, rosmarinic-acid, rutin, saikogenin,saikosaponin, sanguinarine, sciadopitysin, scopoletin, serrapeptase,sinapaldehyde, strophanthidin, syringaldehyde, taraxasterol,taraxasterol-acetate, taspine, tylophorine, umbelliferone, ursolie-acid,and withaferin-a.

Anticapillary-Fragility Agents

Compounds having ‘anticapillary-fragility’ activity may also be includedas an additional ingredient. Capillary-fragility causes telangiectasiaand spider veins, which are preferably reduced or removed from the skinby using an anticapillary-fragility agent. Further, AGES and freeradicals can cause damage to the fine vessels and induce telangiectasiaor spider veins; therefore this class of agents is also useful. Theseagents include, but are not limited to, aescin, aesculetin, aesculetol,aesculin, aesculoside, diosmin, escin, esculetin, esculin, hederagenin,hesperidin, hydroxyethylrutoside, hyperoside, inulicin, luteolol,maniflavone, neoruscogenin, patulin, procyanidin-a-2, quercetol,quercetoside, rhamnetol, ruscogenin, rutin, rutoside, andxanthorhamnoside. Additionally, microcirculation decreases with age,especially around the eyes. Therefore this class is also beneficial andincludes hydroxysuccinimide, chrysin(and other bilirubinolyticsubstances such as gardenin, gardenoside, berberine) or ingredientscontaining such substances eg Haloxyl from Sederma, Nattokinase, andvitamin K in all its forms including menaquinone-7.

Anti-Elastase Agents

Anti-elastase ingredients are also beneficial as additional ingredientswhich enhance the formula's ability to firm the skin. These agentsinclude, but are not limited to, PROTEASYL® TP LS 8657 (fromLaboratories Serobiologiquc), anthocyanins, caffeie-acid, isoquercitrin,procyanidin-a-2, and quercetin. To help target sagging of skin, theseingredients can be included that improve adhesion of cells to thebasement membrane and among themselves by enhancing synthesis of lamininV and integrin alpha 6. One such ingredient is Serilesine from Lipotec.

Alpha Hydroxy Acids

Alpha hydroxy acids and α-hydroxy acid derivatives, known for theirexfoliating and resurfacing properties, may be combined as an ingredientin the topical formulation of the present invention in combination witha saccharide isomerate, green tea, strontium chloride and/or a COX 2inhibitor to prevent stinging. An α-hydroxy acid is an organic compoundcontaining at least one hydroxy group and at least one carboxyl group,and wherein at least one hydroxyl group is located on the α-carbon atom.Typically, the compounds are organic acids having at least onecarboxylic acid group and at least one hydroxyl group on the α-carbonatom, and may contain other functional groups including additionalhydroxyl and carboxylic acid moieties (e.g., glycolic acid, lacticacid).

Liposomes

A In a preferred embodiment, liposomes made by the process described byAGI Dermatics (New York) may be used. These photosomes and ultrasomesare useful in formulations used to target DNA repair associated withphotoaging and are described in U.S. Pat. Nos. 6,623,724 and 6,479,533.A marine-derived photolyase, a DNA-repair enzyme from Anacystis nidulansplankton may be added to the formulation. These enzymes, incorporatedinto a liposome (e.g., Photosome®), are adsorbed through the skin andrepair sun-damaged DNA. Redness and sunburn cell formation may also bereduced or prevented by the addition of these enzymes.

Dermorelaxants

Dermorelaxants, which relax the muscles directly beneath the skin, mayalso be incorporated into the formulation of the present invention.These compounds relax the muscles and reduce wrinkles in the skin.Dermorelaxants include compounds such as myoxinol from cognis, boswelliaextract and hexapeptides (available from Lipotech, Spain) and may beincorporated into the formulation of the present invention. Additionaldermorelaxants include limonoids such as those described in U.S. Pat.No. 6,866,856. Limonoids are plant alkaloids of the Maliaecae family,such as toosendanin and azadirachtin, that are useful for relaxing thefacial expression muscles.

Acetylcholine Precursor

An acetylcholine precursor may be added to the topical formulation ofthe present invention. An acetylcholine recursor is any precursor in thebio-synthetic pathway of acetylcholine, or related pathways. Theseinclude co-factors and precursors of acetylcholine, synthetic enzymesand precursors or enhancers of acetyl production. Useful acetylcholineprecursor compounds for the invention include, but are not limited to,ethylaminoethanol, methylaminoethanol, dimethylaminoethanol,isopropanolamine, triethanolamine, isopropanoldimethylamine,ethylethanolamine, 2-butanolamine, choline, serine, the calcium salt of2-aminoethanol phosphate, the sodium salt of 2-aminoethanol phosphate,the potassium salt of 2-aminoethanol phosphate, and mixtures thereof.Many preferred embodiments employ methylaminoaminoethanol,dimethylaminoethanol, ethylaminoethanol, the calcium salt of2-aminoethanol phosphate, and/or triethanolamine; particularly preferredis dimethylaminoethanol (DMAE) and/or the calcium salt of 2-aminoethanolphosphate.

Additional Agents

Another particularly preferred skin-protective agent is beta glucan,which may be obtained from yeast, oat and mushroom species. It is a freeradical scavenger and stimulates nonspecific immunity.

Hyaluronic acid, a component of connective tissue whose function is tocushion and lubricate the tissue, as well as hyaluronidase inhibitorssuch as extracts of Echinacea species, are also useful as additionalagents in the present formulation.

Forskholin and other agents useful in raising cyclic AMP are alsocontemplated as ingredients in the present formulation.

Lion's mane is included in the formulation in one embodiment. Thismedicinal mushroom (Hericium erinaceum) has been used to treat dementiaand contains at least two classes of compounds, erinacines andhericenones, both which strongly stimulate NGF synthesis (Kawagishi, H.et al., Townsend Letter for Doctors and Patients, 4-2004).

The additional agents can be used in the formulation of the presentinvention, or may be applied separately. The amount of additional agentis dependent upon the activity of that particular agent, and will varydepending upon the preferred formulation. In most instances, each agentwill be present in the formulation in an amount from about 0.001 toabout 30% by weight, preferably from about 0.1 to about 5% by weight,based on the total weight of the preparation.

In some embodiments, certain agents may be included in the formulationin a homeopathically diluted form due to their potential toxicity orirritating potential. Therefore, these agents may be added at much lowerconcentrations. These agents would be formulated according to stricthomeopathic standards and, depending on the substance, the effect may bethe same or different from that of the starting molecule.

Similarly, for highly active agents such as growth factors, the amountof agent added to the formulation may be less than the 0.001% asdescribed above. For these agents, the weight percent may be from0.00001%, or from 0.0001% and up.

Of the additional agents described herein, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more additional agents may be added. The additional agents may becombined with the topical formulation using any combination that one ofordinary skill in the art would perceive to combine. Two or moreadditional agents from one category (e.g., two AGE inhibitors) may beadded to the same formulation. Since the combination of some of theadditional agents may cause adverse effects not outweighed by theirpositive benefit when topically applied to the skin, care will be takenwhen making a topical formulation. Data from the FDA, researchpublications and any other known sources will be used to determine ifthere are known adverse interactions between any of the ATP enhancingagents and additional agents incorporated in the formulation.Combinations with unacceptable adverse effects will not be used in thetopical formulations of the present invention.

IV. Topical Formulations

The formulations of the present invention may also comprisedermatologically acceptable topical carriers. Dermatologicallyacceptable carriers, also known as auxiliaries, are known in the fieldsof dermatology, pharmacology, food technology and related fields. Inparticular, the carriers listed in relevant Pharmacopeia (e.g DAB Ph.Eur. BP NF), and other auxiliaries whose properties do not impedephysiological use when applied to the skin, may be used.

Suitable carriers may be lubricants, wetting agents, emulsifying andsuspending agents, preservatives, anti-irritants, emulsion stabilizers,film formers, gel formers, odor masking agents, resins, hydrocolloids,solvents, solubilizers, neutralizing agents, permeation accelerators,pigments, quaternary ammonium compounds, refatting and superfattingagents, ointment, cream or oil base materials, silicone derivatives,stabilizers, sterilizing agents, propellants, drying agents, opacifiers,thickeners, waxes, emollients, or white oils.

Only effective amounts of macqui berry or macqui berry extract and otheroptional active ingredients are needed. Generally topical application isaccomplished in association with a carrier, and particularly one inwhich the active ingredients are soluble per se or are effectivelysolubilized (e.g., as an emulsion or microemulsion). Where employed, thecarrier is inert in the sense of not bringing about a deactivation oroxidation of the compounds, and in the sense of not bringing about anyadverse effect on the skin areas to which it is applied. In onepreferred practice of the invention, active ingredients are applied inadmixture with a dermatologically acceptable carrier or vehicle so as tofacilitate topical application and, in some cases, provide additionaltherapeutic effects as might be brought about by, e.g., moisturizing ofthe affected skin areas. While the carrier for dermatologicalformulations can consist of a relatively simple solvent or dispersantsuch as water, it is generally preferred that the carrier comprise acomposition more conducive to topical application, and particularly onewhich will form a film or layer on the skin to which it is applied so asto localize the application in areas that will be electricallystimulated and/or aid in the percutaneous delivery of the active agent.

Many preparations of topical carriers are known in the art, and includelotions containing oils and/or alcohols and emollients, vegetable oils,hydrocarbon oils and waxes, silicone oils, animal or marine fats oroils, glyceride derivatives, fatty acids or fatty acid esters oralcohols or alcohol ethers, lecithin, lanolin and derivatives,polyhydric alcohols or esters, wax esters, sterols, phospholipids andthe like, and generally also emulsifiers (nonionic, cationic oranionic), although some of the emollients inherently possess emulsifyingproperties. These same general ingredients can be formulated into acream rather than a lotion, or into gels, or into solid sticks byutilization of different proportions of the ingredients and/or byinclusion of thickening agents such as gums or other forms ofhydrophilic colloids. They may be formulated using conventionaltechniques known to those of ordinary skill in the art.

Additional excipients commonly found in skin care compositions such as,for example, emollients, skin conditioning agents, emulsifying agents,humectants, preservatives, antioxidants, perfumes, chelating agents,buffering agents, etc. may be utilized provided that they are physicallyand chemically compatible with other components of the formulation.Preservatives include, but are not limited to, C₁-C₃ alkyl parabens andphenoxyenthanol, typically present in an amount ranging from about 0.5%to about 2.0% by weight percent, based on the total formulation.Emollients, typically present in amounts ranging from about 0.01% to 5%of the total formulation include, but are not limited to, fatty esters,fatty alcohols, mineral oils, polyether siloxane copolymers, andmixtures thereof. Humectants, typically present in amounts ranging fromabout 0.1% to about 5% by weight of the total formulation include, butare not limited to, polyhydric alcohols such as glycerol, polyalkyleneglycols (e.g., butylene glycol, propylene glycol, dipropylene glycol,polypropylene glycol, and polyethylene glycol) and derivatives thereof,alkylene polyols and their derivatives, sorbitol, hydroxy sorbitol,hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylatedglycerol, propoxylated glycerol, and mixtures thereof. Emulsifiers,typically present in amounts from about 1% to about 10% by weight of theformulation, include, but are not limited to, stearic acid, cetylalcohol, stearyl alcohol, steareth 2, steareth 20, acrylates/C₁₀₋₃₀alkyl acrylate crosspolymers, and mixtures thereof. Chelating agents,typically present in amounts ranging from about 0.01% to about 2% byweight, include, but are not limited to, ethylenediamine tetraaceticacid (EDTA) and derivatives and salts thereof, dihydroxyethyl glycine,tartaric acid, and mixtures thereof. Buffering agents may be added toprovide the formulation with a pH ranging from about 4.5 to about 8.5,more preferably from about 5.5 to about 8.5, most preferably from about6.5 to about 8.0. Typical buffering agents are chemically and physicallystable agents commonly found in cosmetics, and can include compoundsthat are also adjunct ingredients such as citric acid, malic acid, andglycolic acid buffers.

One particular embodiment comprises the use of novel dispersions ofhydrophobes to yield a surfactant-free formulation, by subjecting thematerials to high pressure, high shear processing. Cold processformulations are also a preferred method as they protect certainheat-sensitive sensitive agents in the formulation; they can be obtainedby using self-emulsifying oleosomes such as Natrulon OSF available fromLonza. In one embodiment, the formulation is processed using thecarriers and dry-water process of Aerosil® (Degussa), which is based onfumed silica. (seewww1.sivento.com/wps3/portal/en/aerosil/industries/personal0.html).

According to the invention, the formulations are administered topicallyin the form of a cream, gel, or liquid. The topical administrationprovides the stabilized macqui berry formulation directly to the skin,which is preferably provided with the use of a dermatologicallyacceptable carrier. While the carrier may consist of a relatively simplesolvent or dispersant, such as an oil, it is generally preferred thatthe carrier comprise a material more conducive to topical application,and particularly one which will form a film or layer on the skin towhich it is applied. This localizes the application and provides someresistance to perspiration and/or aids in percutaneous delivery andpenetration of the active ingredients into lipid layers. Many suchcompositions are known in the art, and can take the form of creams,gels, ointments, hydrogels, pastes or plasters, and liquid dosage forms,such as solutions, emulsions, in particular oil-in-water emulsions,suspensions, for example lotions, etc., or even solid sticks. Liposomesor microspheres may also be used.

In some embodiments, the topical formulation will be administered usinga device or method designed to more readily break the skin barrier andprovide the agents in the topical formulation with a faster or moreeffective means through the stratum corneum. These include, for example,oxygen nebulizers and nanosomal mist in conjunction with iontophoresis.A spray or nebulizer may be used to create the nanosomel mist. In oneembodiment, the micro-electronic cosmetic delivery mechanism describedas PowerCosmetics® may be used for delivery of the topical agent to theskin. This method is useful for delivering ionizable compounds to theskin and aids the penetration of small molecules through the stratumcorneum. (www.powerpaper.com).

Only effective amounts of each of the components in the topicalformulation are needed to treat the signs of aging in skin. Generally,an effective amount of the topical formulation is applied to exposed oraffected skin sites in association with a carrier, and particularly onein which the active ingredients are soluble per se or are effectivelysolubilized (e.g. as an emulsion or microemulsion). The term “aneffective amount” of a compound or property means an amount effective inreducing the signs of aging skin, such as wrinkles, pigmentary changes,elastosis, and atrophy. The exact amount required will vary from case tocase, depending on recognized variables such as the compounds employedand the individual subject treated. Thus, it is not possible to specifyan exact “effective amount.” However, an appropriate effective amountmay be determined by one of ordinary skill in the art using only routineexperimentation.

Preferably, the formulation will contain 0.01 to 80% by weight of themacqui berry or macqui berry extract. Desirably, it will contain0.1%-10.0% by weight, or more preferably 1-5% by weight macqui berry ormacqui berry extract. Carriers can be chosen which will solubilize ordisperse the active ingredients at such concentrations as providedherein. One particularly efficacious embodiment contains about 1-5% byweight anthocyanins and 1-3% by weight flavonoid glucuronide in aliposomal carrier.

As used herein, the term “treatment of aging skin” means the treatmentof the symptoms of skin damage due to either chronoaging or photoagingof the skin, which is characterized by wrinkles, loss of elasticity, andhyper-pigmentation. The treatment is effective to enhance the appearanceand/or health of the skin. This includes, for example, reducingoxidative damage in the skin.

The term “effective amount” of a compound or property as provided hereinmeans such an amount as is capable of performing the function of thecompound or property for which an effective amount is expressed. Aspointed out above, the exact amount required will vary from case tocase, depending on recognized variables, such as the compounds employedand the processing conditions observed. Thus, it is not possible tospecify an exact “effective amount.” However, an appropriate effectiveamount may be determined by one of ordinary skill in the art using onlyroutine experimentation.

As used herein, the phrase “formulated for use in a beverage” means thatthe formulation containing the macqui berry or macqui berry extract iscombined with ingredients that are safe for human consumption.Optionally, these ingredients may be to keep the macqui berry or macquiberry extract in solution or to improve or enhance the flavor or color.

As used herein, the phrase “formulated for use in a candle” means thatthe formulation containing the macqui berry or macqui berry extractcontains ingredients to keep the formulation stable within candle wax.The formulation may be adapted to keep the formulation mixed within thecandle wax to maintain a consistent color, or it may provide optimalrelease of aromatics from the macqui berry or additional source toprovide a pleasing smell.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this specification. Each patentdescribed herein is hereby incorporated by reference in its entirety.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

TABLE 2 Ingredient % by weight macqui berry extract 3.0 luteolin7-glucuronide 1.0 carriers and excipients 96.0

Example 2

TABLE 3 Ingredient % by weight macqui berry juice 5.0 quercetin3-(isoferulylglucuronide) 3.0 carriers and excipients 92.0

Example 3

TABLE 4 Ingredient % by weight macqui berry extract 3.0 glutathione 3.0carnosine 1.0 carnitine 1.0 carriers and excipients 92.0

The formulation is encapsulated in a polysaccharide micro-capsule.

Example 4

TABLE 5 Ingredient % by weight macqui berry extract 0.5% bilberryextract 0.5% rosmarinic 0.1% glutathione 1.0% green tea polyphenols 1.0%resveratrol 0.1% phenyl-butyl-nitrone(PBN) 0.1% boldine 0.5% EGF 0.5%rutin 0.5% MMP inhibiting glycosaminoglycans 1.0% tocopherols 0.5% SOD0.5% carriers and excipients 93.2%

Example 5

TABLE 6 Ingredient % by weight macqui berry extract 1.0% rosmarinic 0.5%retionol 1.0% alpha-hydroxy acids 3.0% allantoin 2.0% betaine 5.0%carriers and excipients 87.5%macqui berry extract 1.0% Rosmarinic 0.5% Retinol 1.0% Carriers andExcipients 87.5%

Example 6

TABLE 7 Ingredient % by weight macqui berry extract  0.1% vignaaconitifolia seed extract   5% coffe fruit extract  0.1% saffron extract0.01% betacyanins 0.01% R-lipoic sodium salt 0.01% rosemary extract 0.1% ATP 0.01% EGF 0.01% carnosine 0.01% sodium phytate 0.05% carriersand excipients 93.2%

Having described the invention with reference to particularcompositions, theories of effectiveness, and the like, it will beapparent to those skilled in the art that it is not intended that theinvention be limited by such illustrative embodiments or mechanisms, andthat the modifications can be made without departing from the scope orspirit of the invention, as defined by the appended claims. It isintended that all modifications and variations be included with thescope of the invention. The claims are meant to cover the claimedcomponents and steps in any sequence that is effective to meet theobjectives there intended, unless the context specifically indicates thecontrary.

The invention claimed is:
 1. A topical formulation comprising: macqui berry, a macqui berry extract or a dermatologically acceptable salt thereof containing an effective amount of one or more antioxidants, a stabilizer, and an intracellular glutathione inducer.
 2. The formulation of claim 1, wherein the stabilizer is a glucuronide, a glycuronide, diethylhexyl syringylidene malonate, a microencapsulation agent, or a light and/or air-blocking packaging.
 3. The formulation of claim 2, wherein the microencapsulation agent is a liposome or a nanosome.
 4. The formulation of claim 2, wherein the microencapsulation agent is a biodegradable substance.
 5. The formulation of claim 2, wherein the light-blocking packaging blocks light between 450 nm and 750 nm.
 6. The formulation claim 1, which comprises a macqui berry, macqui berry extract, in an amount of from about 0.05 to 50% by weight, based on the total weight of the formulation.
 7. The formulation claim 1, further comprising an additional agent.
 8. The formulation of claim 7, wherein the additional agent is an antioxidant.
 9. The formulation of claim 8, wherein the additional antioxidant is an antioxidant endogenous to the human body.
 10. The formulation claim 8, wherein the additional antioxidant is selected from the group consisting of grape seed procyanidins, tocopherols, tocotrienols, astaxanthin, lutein, lycopene, phytofluene, phytoene, rubixanthin, beta-cryptoxanthin, zeaxanthin, tumeric extract, bilberry anthocyanins, betacyanins, a purified single anthocyanin or combination of selected purified individual anthocyanins, black raspberry extract, blueberry anthocyanins, acai extract, aronia extract, prune extract, pomegranate extract, ellagic acid, honokiol, magnolol, silymarin, quercetin, naringenin, rosmarinic acid, rosemary extract, saffron extract, clove essential oil, wolfberry extract, noni fruit extract, phyllantus emblica extract, camellia sinensis extract, epicatechin-gallate, Garcinia mangostana extract, shikimic acid, montmorency cherry extract, anthocyanin-containing extracts of red, blue, magenta, purple or black flowers, whole coffee fruit extract, chlorogenic acid, caffeic acid, ferulic acid, olive extract, and hydroxytyrosol.
 11. The formulation of claim 10, wherein the formulation comprises a metal chelating agent.
 12. The formulation of claim 8, wherein the antioxidant is formulated in a ratio with the macqui berry or macqui berry extract wherein the ORAC value is optimized.
 13. The formulation claim 8, wherein the additional antioxidant is a betacyanin.
 14. The formulation claim 1, further comprising an AGE inhibitor, a collagen enhancing agent, a mitochondrial resuscitant, a sunscreen agent, an anti-edemic agent, a collagenase-inhibitor, an anti-inflammatory agent, a phenylpropanoid glycoside, a depigmenting agent or agent addressing hyperpigmentation, a skin-protective lipids, hyaluronic acid, an alpha hydroxy acid, an agent useful for treating hormonal decline, an anti-acne agent, an agent altering lipolytic activity, an anti-cellulitic agent, an agent altering anti-capillary-fragility, an anti-elastase agent, an anti-erythema agent, an agent that raises cyclic AMP, or nicotinamide-adenine-dinucleotide.
 15. A method of treating skin, said method comprising administering a topical formulation comprising: macqui berry, a macqui berry extract or a dermatologically acceptable salt thereof containing an effective amount of one or more antioxidants, a stabilizer, and an intracellular glutathione inducer.
 16. The method of claim 15, wherein the stabilizer is a glucuronide, a glycuronide, a diethylhexyl syringylidene malonate, microencapsulation agent, or a light and/or air-blocking packaging.
 17. The method of claim 16, wherein the microencapsulation agent a liposome or a nanosome.
 18. The method of claim 16, wherein the light-blocking packaging blocks light between 450 nm and 750 nm. 